Automated protein blotting components and systems

Abstract

Systems, methods, and components for automated processing and analyzing a biological sample are disclosed herein. Such components include pre-filled gel cards, gel card cassettes and blotting cartridges for use in automated electrophoresis and protein transfer. Systems includes automated electrophoresis and transfer modules that automatically separate proteins from the biological sample into a plurality of bands and transfer the plurality of bands to a membrane using the gel card cassettes and blotting cartridges herein. Compared to conventional methods, the disclosed system and method provide improvements in reduced experiment time, reduced error, improved precision, and reduced consumption of consumables and chemicals.

Description

KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO AUTOMATED PROTEIN BLOTTING COMPONENTS AND SYSTEMS CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This PCT application claims the benefit of priority to U.S. Provisional Application No.63 / 685,142 filed August 20, 2024, the entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION

[0002] This invention relates to apparatuses and methods for processing biomolecules. BACKGROUND

[0003] Protein blotting is a widely used analytical immunoblotting technique to detect target proteins in a sample. The workflow typically includes collecting and preparing a sample (such as cell extract), separating the proteins in a sample, transferring the separated proteins from a gel to a membrane, probing the membrane with antibodies that target the proteins of interest, using chemiluminescent or fluorescent imaging to detect the target proteins, and quantifying the resulting chemiluminescent or fluorescent signals. Moreover, there are several different variations of protein blotting technique that follow a similar workflow such as Western blotting, Eastern blotting, Northwestern blotting, Southwestern blotting, Far Western blotting, and variants thereof.

[0004] Although these analytical blotting methods are commonly used, its workflow is time consuming and requires many manual steps (e.g., sample preparation, electrophoresis, transfer, immunodetection, image acquisition, and image analysis), which, in total, may take one to two days to perform. With increasing manual steps, the probability of error is compounded. When an error is detected late in the protein blotting protocol, the time and effort spent performing the protein blot can be lost, often requiring repeating the experiment. Moreover, conventional protein blotting techniques require considerable amounts of expensive antibodies for processing, increasing the impact of the error. Accordingly, there is a need for an improved protein blotting method that decreases the time to results, improves reproducibility, lessens the chance for errors, and reduces antibody consumption.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO SUMMARY

[0005] In one aspect, the disclosed technology relates to an automated system for analyzing a biological sample, the system including: (i) a separation module comprising: (a) a gel disposed on a support layer, wherein the gel comprises a plurality of microwells, and wherein the gel is permeable to an electric field along a perpendicular axis at least within a sample separation flow path from a sample well to an opposite end of the gel; (b) a set of anode and cathode buffer reservoirs comprising running buffer; (c) one or more electrode(s) embedded in the cathode and anode buffer reservoir; and (ii) a blotting module comprising: (a) a blotting cartridge (also referred to as a “membrane cartridge”) comprising: a chamber comprising a first opening on a first side and a second opening on a second side; a transfer membrane attached directly or indirectly to the chamber and spanning the first opening of the blotting cartridge, vacuum / pressure connection to the second opening; and an anode electrode, wherein the blotting cartridge is movable between the blotting module and an upper surface of the gel in the separation module; (b) a blotting reservoir positioned under the separation module to receive transfer buffer; (c) a reagent tray; and an image sensor positioned below the blotting cartridge, wherein the image sensor is optionally in contact with the transfer membrane during imaging. In some embodiments, the imaging module further comprises an excitation source positioned below the blotting cartridge, wherein the excitation source is configured to excite biological samples on the transfer membrane; and optionally, a filter positioned between the image sensor and the blotting cartridge, wherein the filter corresponds to a wavelength of the excitation source.

[0006] In some embodiments, the gel comprises agarose, polyacrylamide, or a blend thereof. In some embodiments, the gel is polyacrylamide. In some embodiments, the blotting cartridge comprises a support layer backing the transfer membrane. In some embodiments, the blotting reservoir contains a cathode electrode. In some embodiments, the microwells have a width of about 0.5 mm to about 5 mm and have a depth of about 0.3 to about 10 mm. In some embodiments, the system further comprises a data visualization module, which comprises a computer system for analyzing emission signals from the transfer membrane. In some embodiments, the gel comprises about 4% to about 20% of acrylamide. In some embodiments, the gel comprises a label. In some embodiments, the label is a haloalkane selected from the group consisting of trichloroethanol, chloroform, trichloroacetic acid, trichloroethane, bromoform, and iodoacetic acid; or a UV excitable dye.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0007] In some embodiments, the support layer of the blotting cartridge comprises a porous material. In some embodiments, the porous material is selected from the group consisting of polyethylene, polypropylene, cellulose, porous plastic polymers, cellulose / polymer blend, glass fibers, porous ceramics, graphite, aluminum oxide, porous foams, and combinations thereof. In some embodiments, the gel is supported between two porous films.

[0008] In some embodiments, the system separates, transfers, and detects proteins in the biological sample in less than about 2 hours. In some embodiments, the transfer membrane comprises a material selected from polyvinylidene fluoride (PVDF), nylon and nitrocellulose. In some embodiments where PVDF membrane is used it may be made hydrophilic prior to attachment to the cartridge, in this way the instrument does not need to perform wetting with alcohol and equilibration with buffer prior to use saving time in the protocol. The membrane can be made hydrophilic by applying coatings or by incorporating detergents such as SDS which is compatible with the process. In some embodiments, the anode in the blotting cartridge reservoir is electrically paired with a cathode in the blotting tray reservoir. In some embodiments, the reagent tray comprises compartments pre-filled with blotting reagents. In some embodiments, the blotting reagents are independently selected from blocking buffer, wash buffer, primary antibody solution, primary antibody diluent, secondary antibody solution, secondary antibody diluent, diluent, protein-conjugated reporter molecule, oligonucleotide- conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution. In some embodiments, the compartments are each pre-filled with about 1 mL to about 10 mL of the blotting reagents. In some embodiments, the blotting cartridge is connected to a vacuum / pressure source and delivers each blotting reagent to the membrane when the membrane is in contact with corresponding compartments of the reagent tray. In some embodiments, the system further comprises a waste module coupled to the vacuum source.

[0009] In some embodiments, the excitation source comprises at least one light emitting diode (LED) emitting light at a wavelength in a range from about 325 nm to about 700 nm, an ultraviolet light source, or both. In some embodiments, the image sensor is configured for colorimetric, chemiluminescent, or fluorescent detection. In some embodiments, the system further comprises an XYZ gantry or robot attached to the blotting cartridge for controlling movement of the blotting cartridge. In some embodiments the blotting cartridge is attached to gantry or robotic arm that moves the cartridge in the z dimension and a stage in the instrument carrying the gel, reagent tray and camera move in along an XY plane.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0010] In another aspect, the disclosed technology further relates to a method for detecting proteins in a biological sample, the method comprising: (a) inserting the gel into a blotting reservoir, wherein the gel is optionally pre-loaded with the biological sample, molecular weight standard, and running buffer; (b) engaging a separation anode with a separation cathode in the separation module, and applying an electric current between the separation anode and cathode to separate the proteins into a plurality of bands; (c) dispensing transfer buffer to the blotting tray reservoir under the gel and into the blotting cartridge; (d) lowering the blotting cartridge toward the first surface of the gel until the transfer membrane contacts the first surface of the gel, engaging a transfer anode with a transfer cathode, and applying an electric current between the transfer anode and the transfer cathode to transfer the plurality of bands from the gel to the transfer membrane; (e) removing the transfer buffer from the blotting cartridge and washing the blotting cartridge with water or a wash buffer; (f) processing the transfer membrane by sequentially aspirating one or more blotting reagents from one or more individual compartments of the reagent tray through the transfer membrane of the blotting cartridge to form a plurality of labeled bands on the transfer membrane; and (g) detecting an optical signal from each of the plurality of labeled bands.

[0011] In some embodiments, the gel is a Stain-FreeTM gel, the plurality of labeled bands are a plurality of labeled protein bands and the method further comprises performing Stain- FreeTM imaging of the gel, wherein after step (b), the Stain-FreeTM gel comprising a plurality of protein bands is exposed to ultraviolet light to form labeled protein bands. In some embodiments, steps (b) through (g) are automated. In some embodiments, the method comprises reporting results in tabular and / or graphical formats. In some embodiments, the method comprises normalizing the optical signal. In some embodiments, the optical signal is a colorimetric, fluorescent, or chemiluminescent signal. In some embodiments, the separation in step (b) occurs in about 5 minutes to about 20 minutes. In some embodiments, step (d) comprises lowering the blotting cartridge to the upper surface of the gel at a pressure from about 0.5 kg-force to about 2 kg-force. In some embodiments, the transferring the plurality of bands in step (d) occurs in about 1 minute to about 10 minutes. In some embodiments, the removing the transfer buffer from the blotting cartridge in step (e) is carried out by vacuum aspiration or pressure. In some embodiments, the washing the blotting cartridge in step (e) is performed at a water or buffer wash station, wherein the blotting cartridge is rinsed and aspirated to remove excess transfer buffer solution from the transfer membrane. In some embodiments, the blotting cartridge is rinsed after aspirating each of the blotting reagents inKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO the reagent tray in step (f). In some embodiments, the one or more blotting reagents are selected from blocking buffer, primary antibody, primary antibody diluent, wash buffer, secondary antibody, secondary antibody diluent, diluent, protein-conjugated reporter molecule, oligonucleotide-conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution.

[0012] Yet, in another aspect, the disclosed technology relates to A kit for automated analysis of proteins in a biological sample, the kit comprising: (i) one or more components selected from: (a) a precast gel; (b) a disposable blotting cartridge; (c) a reagent tray; (d) a running buffer solution; (e) a transfer buffer solution; and (f) a cleaning pad; and (ii) packaging information and instructions for use, wherein the precast gel comprises agarose, polyacrylamide, or a blend thereof. In some embodiments, the reagent tray comprises compartments pre-filled with one or more components selected from blocking buffer, wash buffer, primary antibody solution, primary antibody diluent, secondary antibody solution, secondary antibody diluent, diluent, protein-conjugated reporter molecule, oligonucleotide- conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution. In some embodiments, the precast gel has a length of about 2 cm to about 8 cm and has a width of about 2 cm to about 10 cm. In some embodiments, the disposable blotting cartridge comprises a transfer membrane attached directly or indirectly to and spanning a first opening of a chamber in the blotting cartridge. In some embodiments, the transfer membrane is polyvinylidene fluoride (PVDF), nylon, or nitrocellulose. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG.1 is a block diagram of a system for analyzing biological samples, according to the present disclosure.

[0014] FIG. 2A is a perspective view of one embodiment of a gel assembly of the present disclosure.

[0015] FIG. 2B is an enlarged view of one embodiment of a gel assembly of the present disclosure.

[0016] FIG.3A is a perspective view of another embodiment of a gel assembly of the present disclosure and a corresponding sample separation path (inset).

[0017] FIG.3B is a perspective view of another embodiment of a gel assembly of the present disclosure and a corresponding sample cup-loading device (inset).KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0018] FIG. 4 is a side perspective view of another embodiment of a gel assembly of the present disclosure positioned within a blotting tray.

[0019] FIG.5 is a perspective view of one embodiment of a blotting cartridge of the present disclosure.

[0020] FIG. 6 is a perspective view of another embodiment of a blotting cartridge of the present disclosure.

[0021] FIG. 7 is a side-perspective view of another embodiment of a blotting cartridge of the present disclosure.

[0022] FIG. 8A is a perspective view of another embodiment of a blotting cartridge of the present disclosure in a disassembled configuration.

[0023] FIG. 8B is an assembled perspective view of an embodiment of a blotting cartridge of FIG.8a in an assembled configuration.

[0024] FIG. 9 is a perspective view of a protein separation module and transfer module in an operating configuration according to the present disclosure.

[0025] FIG.10 is a perspective view of another embodiment of a protein separation module and transfer module in an operating configuration according to the present disclosure.

[0026] FIGS. 11A-11B depicts an embodiment of a reagent tray of the present disclosure with a blotting cartridge dipped into one of the tray sections.

[0027] FIG.12A depicts an embodiment of an imaging module of the present disclosure.

[0028] FIG. 12B depicts another embodiment of an imaging module of the present disclosure.

[0029] FIG. 13 depicts a flow diagram of an illustrative method for analyzing biological samples.

[0030] FIG. 14 depicts an exemplary consumable kit that may be used with a system and method of the present disclosure.

[0031] FIGS. 15A-15C show a U-cast cassette and gel card in accordance with some embodiments.

[0032] FIGS. 16A-16B show an ion flow dam to facilitate electrophoresis and a U-cast cassette and gel card with two ion flow dams, in accordance with some embodiments.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0033] FIG.17 shows a gel card with a connector to facilitate removal from the cassette, in accordance with some embodiments.

[0034] FIGS. 18A-18C show an exemplary gel card design, in accordance with some embodiments.

[0035] FIGS.19A-19D show an electrophoresis and transfer box, in accordance with some embodiments.

[0036] FIG.20A-21B demonstrate the results of the above described methods, in accordance with some embodiments.

[0037] FIGS.22A-22C shows views of an exemplary gel card cassette with integrated buffer reservoirs, in accordance with some embodiments.

[0038] FIG.23 shows a high density gel card, in accordance with some embodiments.

[0039] FIG. 24 shows an S-curve design of gel card cartridge, in accordance with some embodiments.

[0040] FIGS. 25A-25B show an exemplary membrane cartridge with wire electrode, in accordance with some embodiments.

[0041] FIG. 26 shows a membrane cartridge with integrated plunger, in accordance with some embodiments.

[0042] FIGS. 27-28 show variations of blotting cartridges, in accordance with some embodiments.

[0043] FIG. 28A shows a fully assembled embodiment of membrane cartridge, in accordance with some embodiments.

[0044] FIGS. 29A-29C shows different views of an embodiment 3400 of a two-piece blotting cartridge, in accordance with some embodiments.

[0045] FIG. 30 shows another blotting cartridge with membrane, in accordance with some embodiments.

[0046] FIG.31 shows an electroelution cartridge, in accordance with some embodiments.

[0047] FIG.32 shows a system overview that includes a filtration station, in accordance with some embodiments.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0048]

[0049] FIG. 33 shows an exemplary automated workflow, in accordance with some embodiments.

[0050] FIG.34 shows the Western blot results, in accordance with some embodiments. DETAILED DESCRIPTION

[0051] This disclosure relates to blotting systems and methods of automatically detecting and analyzing proteins in a biological sample. The systems described herein enable decreased time to results, can improve blotting consistency and reproducibility, and reduces the amount of antibody used. The systems can be used for analytical blotting methods such as Western blotting, Eastern blotting, Northwestern blotting, Southwestern blotting, Far Western blotting, and variants thereof.

[0052] In some embodiments, the disclosed system may be used for a Western blotting workflow and may include collecting and preparing a sample (such as cell extract), separating the proteins in a sample via gel electrophoresis, transferring the separated proteins from a gel to a membrane, probing the membrane with antibodies that target the proteins of interest, using chemiluminescent or fluorescent imaging to detect the target proteins, and quantifying the resulting chemiluminescent or fluorescent signals. In other blotting techniques such as, far Western blotting, rather than using an antibody for detection of a target protein in a blot, the method identifies proteins on the blot due to the presence or absence of binding sites for a non- antibody protein probe molecule, thus characterizing specific protein-protein interactions.

[0053] In another embodiments, the disclosed system may be used for an Eastern blotting workflow. In Eastern blotting, the proteins are separated and transferred to a membrane in the same fashion as in Western blotting, but in this method, post translation modifications (PTM) of the proteins are detected. In some embodiments, specific antibodies or binders to the modification or PTM are used for detection; for example, antibodies against phosphorylation sites, or lectins which bind to carbohydrate moieties decorating the protein of interest. These PTM binders may be conjugated with a detectable label (fluor, HRP, AP, biotin, etc) or detected using a secondary labeled binding reagent analogous to a labeled secondary antibody. For certain embodiments of the present disclosure the reagent tray would have sections that contain one or more PTM binders that contain a detectable label or can be detected by a secondaryKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO reagent in the tray that recognizes the PTM binder. Thus, by adjusting the steps used to aspirate the solutions in the reagent tray the Eastern blot method may be performed.

[0054] In some embodiments, two additional variants of Western blotting techniques, Southwestern blotting and Northwestern blotting may be encompassed by the system of the present disclosure. In these methods, the proteins may be first separated and transferred to a membrane in the same fashion as in Western blotting, but target proteins are identified not by an antibody, but by whether they bind to a particular DNA (Southwestern) or RNA (Northwestern). Accordingly, these methods are directed to detecting specific DNA or RNA binding proteins on the blot. These DNA or RNA oligonucleotides can carry a label or tag that can be detected directly (fluorescent molecule) or indirectly e.g. digoxigenin using an anti-DIG antibody or biotin with a labeled streptavidin. By adjusting the steps and solutions in the reagent tray to aspirate the solutions across the membrane on the blotting cartridge (also referred to as a “membrane cartridge”), the Southwestern and Northwestern blot methods may further be performed using the method and system of the present invention.

[0055] In another aspect, the instrument described herein could be used for nucleic acid separations. Besides western blotting and its variants, this device and gel cards could also be used to perform electrophoresis of nucleic acids to separate by size. Gel cards can be cast with agarose and electrophoresis performed using TBE or TAE buffer containing an intercalating fluorescent dye for detection. The size of analyzed fragments would be compared to DNA or RNA sizing ladders. The instrument would then image the gel card. The membrane cartridge containing for example a charged nylon membrane can then be used to transfer the nucleic acids from the gel card to the membrane analogous to the step in Western blotting. Afterwards Southern and Northern blotting detection can be performed using appropriate solutions in a reagent tray specific to that application. The Nucleic acid sizing application is widely performed by users especially for next-generation sequencing sample preparation protocols. Since the membrane cartridge contains an electrode, it can be adapted to perform electroelution (see FIGS.31A-31C) and recovery of specifically sized DNA / RNA fragments for downstream use. In this sense, the device can also perform as a sample preparation device. Overview of System

[0056] With reference now to FIG. 1, a block diagram of a system 100 for analyzing biological samples is depicted. The system 100 automatically processes biological samples, including separating proteins in the biological samples by electrophoresis to form a pluralityKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO of protein bands, blotting or transfer of the plurality of protein bands to a transfer membrane, processing the transferred plurality of protein bands for immunodetection, acquiring quantitative and qualitative signals, and analyzing the detected data. System 100 may comprise a manual component for preparing the biological samples for analysis and an automated component for the separation, transfer, and imaging of the plurality of protein bands. In some embodiments, the transfer occurs using Trans-Blot® Turbo™ (TBT) (Bio-Rad) transfer conditions. In some embodiments, the system may also be configured for an automated Stain- FreeTMmodule. In some embodiments, the automated component may be contained in and / or mounted within a housing. The manual component may include the user loading biological samples onto a suitable medium, i.e., a gel, for separation of proteins in the biological samples by electrophoresis, as seen in block 101. In some embodiments, the user may also load consumables i.e., gel, blotting cartridge, transfer membrane, buffer solutions, into the device first, then the system may then automatically load the running buffer and then the samples into the wells of the gel. The samples may be preheated to denature the protein and dispensed from the tubes in the heat block to the wells in the gel. The manual component may comprise computer system 102 configured to create, modify, and upload protocol script(s) 103 to automated component by input communication path 105 that describe various experimental parameters including, but not limited to, electrophoresis voltage, transfer voltage, separation time, transfer time, volume of solution aspirated, aspiration rates, incubation periods, type of detection, excitation parameter, detection parameters, imaging parameters, and data output parameters.

[0057] In some embodiments, computer system 102 provides information to a user and receives inputs from a user. This process can include, for example, providing information to a user via a user interface and / or receiving user inputs via the user interface. In some such embodiments, computer system 102 may include one or several hardware features that provide information to the user, such as, for example, one or several screens, speakers, displays, or the like. In some embodiments, the computer may include one or several hardware features that receive user inputs, such as, for example, one or several keyboards, keypads, mouse, microphones, cameras, or the like. In some embodiments, computer system 102 may be hooked to another computing device, and computer system 102 may provide information to this other computing device and can receive user inputs from this other computing device.

[0058] In some embodiments, computer system 102 may comprise one or several computing devices, which can include, for example, one or several personal computers, laptops,KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO computing devices, tablets, smartphones, smart devices, or the like. In some embodiments, the computer can comprise at least a processor and memory. The memory may comprise stored instructions in the form of computer code or a script that, when executed by the processor, causes the computer to take one or several actions. The memory can comprise primary and / or secondary memory. The memory can include, for example, cache memory, RAM, ROM, PROM, EPROM, EEPROM, one or several solid-state drives (SSD), one or several hard drives or hard disk drives, or the like. Thus, in some embodiments, the memory can include volatile and / or non-volatile memory. The processor may include one or several microprocessors, such as one or several Central Processing Units (CPUs) and / or one or several Graphics Processing Units (GPUs). The processor can be a commercially available microprocessor from Intel®, Advanced Micro Devices, Inc.®, Nvidia Corporation ®, or the like. In some embodiments, the computer system or device may interface with “cloud-based” computing resources and services, such as Amazon Web Services (AWS), Microsoft Azure, Google Cloud Platform, IBM Cloud, and the like, where system access, control, data transfer, storage, and processing can occur.

[0059] Once the biological samples have been prepared and the experimental protocol script uploaded, the automated component performs the separation, optional Stain-FreeTM activation and imaging, blotting / transfer, blot immuno-processing, and imaging of the separated and probed samples. The automated component may comprise separation module 110, which separates the proteins in the biological samples loaded onto gel 101 by electrophoresis and may comprise cathode 111, anode 112, and running buffer 113 stored in cathode and anode buffer reservoirs. It should be understood that any gel for processing of biological samples may be used, including but not limited to polyacrylamide gel, agarose gel, or a blend thereof. After separating the proteins by molecular weight and producing protein bands along gel, automated component may comprise Stain-FreeTMmodule 120 where the separated proteins are imaged before transferring the proteins to a blotting module 130. In some embodiments, Stain-FreeTMmodule 120 may comprise an ultraviolet (UV) source 121 to activate the gel and an imaging sensor 122 to collect the signals. In some embodiments, Stain-FreeTMmodule 120 may use a same sensor / detector as imaging module 140. In some embodiments, the Stain-FreeTMmodule 120 may comprise a separate camera system, i.e., charged-coupled device (CCD), or complementary metal-oxide semiconductor (CMOS) device and the like, located directly above the gel for UV-activation and imaging of the gel. Further details regarding Stain-FreeTMimaging are illustrated and described, for example, in U.S. Patent No. 9,606,111 to Freeby etKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO al. the entire contents of which are incorporated herein by reference. In some embodiments, blotting module 130 transfers protein bands from the gel 101 onto transfer membrane 131. In some embodiments, blotting module 130 may comprise blotting cartridge 132 that is attached to transfer membrane 131 either directly or indirectly, blotting tray 133 that holds gel 101, and transfer buffer 134. In some embodiments, the cartridge can vibrate or shake following the transfer. In some embodiments, blotting module 130 may comprise anode 135 for protein transfer or blotting, connected to the blotting cartridge and cathode 136 connected to the blotting tray 133, providing an electric potential path for driving electric current between gel 101 and blotting cartridge 132 for transferring the separated proteins from gel 101 to transfer membrane 131. After transferring, transfer membrane 131 is rinsed at wash station 137. In some embodiments, a second blotting cartridge can be used in place of the blotting tray.

[0060] In some embodiments, the buffer reservoirs are built into the separation module as part of the molding and / or assembly of the part. In some embodiments, the sample wells and / or reservoirs are placed onto an open-face gel where the bottom rim of each well penetrates into the gel layer sealing the edge; this method of sample loading is sometimes referred to as cup- loading. In further embodiments, the buffer reservoirs are separate reservoirs similar to those used in a DNA sub-cell gel box, where the gel is placed on a platform that spans between the reservoirs.

[0061] In some embodiments, blotting module 130 may comprise reagent tray 139 that contains various components for processing the transferred proteins for immunodetection, including, but not limited to, blocking buffer, wash buffer, primary antibody solution, primary antibody diluent, secondary antibody solution, secondary antibody diluent, diluent, protein- conjugated reporter molecule, oligonucleotide-conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution. In some embodiments, blotting cartridge 132, now comprising the transferred protein bands on transfer membrane 131, is automatically introduced to components in reagent tray 139 and automatically routed to imaging module 140 for detection of relevant proteins.

[0062] In some embodiments, imaging module 140 may include illumination / excitation source 141, optical filter 142, and image sensor 143 configured for detection of colorimetric detection, chemiluminescence detection, fluorescence detection, as well as Stain-FreeTMimaging. In some embodiments, the illumination / excitation source 141 may include at least one light-emitting diode (LED) emitting light at a wavelength in the Ultraviolet-visible (UV-KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO Vis) range from about 100 nm to about 700 nm. In some embodiments, LEDs may emit light at a wavelength in ultraviolet range from about 100 nm to about 325 nm. In some embodiments, LEDs may emit light at a wavelength in the visible range from about 325 nm to about 700 nm. In some embodiments, LEDs may emit light at a wavelength in the near IR range from about 700 nm to about 1000 nm. In some embodiments, excitation source 141 may comprise monochromatic laser emitting single wavelength between about 100 nm and about 700 nm, between about 100 nm and about 200 nm, between about 200 nm and about 300 nm, between about 300 nm and about 400 nm, between about 400 nm and about 500 nm, between about 500 nm and about 600 nm, between about 600 nm and about 700 nm, or may be any other or intermediate wavelength and / or range of wavelengths. In some embodiments, illumination / excitation source 141 may include at least one light source that emits visible (from about 325 nm to about 700 nm), ultraviolet (from about 100 nm to about 325 nm), or near- infrared (NIR) to infrared (IR) (from about 700 nm to about 1000 nm) light. Further details regarding the receiver mechanisms are illustrated and described, for example, in U.S. Patent Publication Nos. 2023 / 0199130 to Swihart et al., 2021 / 0356397 to Swihart et al., and U.S. Patent Application No. 18 / 594,870 to McDonald et al., the entire contents of which are incorporated herein by reference.

[0063] After imaging and / or detection of the transferred proteins in imaging module 140, data is converted into graphical, optical, and tabular data and sent to data collection 104 of computer system 102 through output communication path 144, thereby completing the automated processing and analysis of biological samples. In some embodiments, cleaning pad 138 may be snapped on, or press-fit over the surface of transfer membrane 131 and used to clean image sensor 143 or camera or surface above a camera and disposed along with blotting cartridge 132 after imaging and / or detection of proteins and disposed at end of the protocol.

[0064] In some embodiments, the process or time to results (i.e., the separation, transfer, processing, and detection of proteins in the biological samples) is achieved in less than about 5 hours. In some embodiments, the process occurs in less than about 4.5 hours, less than about 4 hours, less than about 3.5 hours, less than about 3 hours, less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, about 1 hour, or any time in-between. In one embodiment, the process occurs from about 1 to about 2 hours.

[0065] With reference now to FIGS. 2A and 2B, a perspective view (FIG. 2A) and an enlarged view (FIG.2B) of an embodiment of a precast gel assembly 200 are shown. As seenKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO in FIG. 2A, a precast gel assembly 200 may include a gel 201 disposed on a cassette 210 holding the gel 201 in place. In some embodiments, gel 201 may be a gel. In some embodiments, gel may comprise about 4% to about 20%, about 4% to about 6%, about 6% to about 8%, about 8% to about 10%, about 10% to about 12%, about 12% to about 14%, about 14% to about 16%, about 16% to about 18%, about 18% to about 20%, about 4% to about 18%, about 6% to about 16%, about 8% to about 14%, about 12% to about 10% of acrylamide. In some embodiments, gel 201 may comprise a gradient from about 4% to about 20% of acrylamide, or any range in between. In some embodiments, a precast gel 201 may be about 2 cm to about 8 cm in length and about 2 cm to about 10 cm in width.

[0066] In some embodiments, the gel 201 may comprise a label capable of emitting a signal when illuminated with ultraviolet light. In some embodiments, the label may include but is not limited to, haloalkane that interacts with tryptophan residues in one or more proteins separated into protein bands in the gel to form labeled protein bands and the haloalkane is selected from the group consisting of trichloroethanol, chloroform, trichloroacetic acid, trichloroethane. In some embodiments, the label is selected from the group consisting of bromoform, iodoacetic acid, an ultraviolet (UV) excitable dye, Alexa Fluor™ 350 and other coumarin derivatives, Pacific Orange™ dye, Cascade® Blue and other pyrene derivatives, Cascade Yellow and other pyridyloxazole derivatives, dansyl chloride, dapoxyl dye, and bimane. Further details of UV- excitable labels are illustrated and described, for example in U.S. Patent No. 10,732,145 to McKee et al., the entire contents of which are incorporated herein by reference. One example of a UV-excitable label for nucleic acids is ethidium bromide.

[0067] In some embodiments, a precast gel assembly 200 may comprise a plurality of microwells 203 into which biological samples are loaded. In some embodiments, a precast gel assembly 200 may comprise from 1 to 20 microwells, from 1 to 5 microwells, from 5 to 10 microwells, from 10 to 15 microwells, from 15 to 20 microwells, from 1 to 18 microwells, from 2 to 16 microwells, from 4 to 14 microwells, from 6 to 12 microwells, or from 6 to 10 microwells. In some embodiments, each microwell 203 may be about 0.05 mm to about 0.1 mm in width, about 0.1 mm to about 0.5 mm, about 0.5 mm to about 1 mm, about 1 mm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, about 2.5 mm to about 3 mm, about 3 mm to about 3.5 mm, about 3.5 mm to about 4 mm, about 4 mm to about 4.5 mm, about 4.5 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.5 mm to about 5 mm, about 1 mm to about 4.5 mm, about 1.5 mm to about 4 mm, about 2 mm to about 3.5, or about 2.5 mm to about 3.0 mm.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0068] In some embodiments, each microwell 203 may be about 0.05 mm to about 0.1 mm in length, about 0.1 mm to about 0.5 mm, about 0.5 mm to about 1 mm, about 1 mm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, about 2.5 mm to about 3 mm, about 3 mm to about 3.5 mm, about 3.5 mm to about 4 mm, about 4 mm to about 4.5 mm, about 4.5 mm to about 5 mm, about 0.05 mm to about 5 mm, about 0.5 mm to about 5 mm, about 1 mm to about 4.5 mm, about 1.5 mm to about 4 mm, about 2 mm to about 3.5, or about 2.5 mm to about 3.0 mm.

[0069] In some embodiments, each microwell 203 may be about 0.1 mm to about 10 mm in depth, about 0.1 mm to about 0.5 mm, about 0.5 mm to about 1 mm, about 1 mm to about 1.5 mm, about 1.5 mm to about 2 mm, about 2 mm to about 2.5 mm, about 2.5 mm to about 3 mm, about 3 mm to about 3.5 mm, about 3.5 mm to about 4 mm, about 4 mm to about 4.5 mm, about 4.5 mm to about 5 mm, about 5 mm to about 5.5 mm, about 5.5 mm to about 6.0 mm, about 6.5 mm to about 7 mm, about 7 mm to about 7.5 mm, about 7.5 to about 8 mm, about 8 mm to about 8.5 mm, about 8.5 mm to about 9 mm, about 9 mm to about 9.5 mm, or about 9.5 mm to about 10 mm.

[0070] In some embodiments, the capacity of each microwell 203 is about 1 µl to about 50 µl, about 5 µl to about 50 µl, about 10 µl to about 45 µl, about 15 µl to about 40 µl, about 20 µl to about 45 µl, about 25 µl to about 40 µl, about 30 µl to about 45 µl, about 35 µl to about 40 µl, or about 1 µl to about 5 µl, about 5 µl to about 10 µl, about 10 µl to about 15 µl, about 15 µl to about 20 µl, about 20 µl to about 25 µl, about 25 µl to about 30 µl, about 30 µl to about 35 µl, about 35 µl to about 40 µl, or about 45 µl to about 50 µl. In some embodiments, for single cell microwell blotting application, the capacity may be from about 1 nl to about 50 nl, about 5 nl to about 50 nl, about 10 nl to about 45 nl, about 15 nl to about 40 nl, or about 20 nl to about 35 nl, about 25 nl to about 30 nl.

[0071] In some embodiments, a gel assembly 200 may comprise a cathode reservoir 207 and an anode reservoir 209 that houses a negatively charged cathode and a positively charged anode, respectively. In some embodiments, the electrodes are integrated into the reservoirs, while in other embodiments the system positions the electrodes into the buffer reservoirs before electrophoresis. In some embodiments, the gel cassette 210 may comprise one or more cassette feet 204 to create a gap beneath the protein separation region and where buffer or a wetted blot stack may be positioned to provide protein transfer in the z-direction. In some embodiments, running buffer is disposed at cathode reservoir 207 and anode reservoir 209 such that when anKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO electric field is generated between cathode reservoir 207 and anode reservoir 209, protein sample 211 is separated as protein bands 213 along separation path 205 from microwells 203 toward anode reservoir 209 based on the molecular weight of the protein as seen in FIG. 2B with smaller proteins displaced closer to the anode reservoir 209.

[0072] In some embodiments, the gel assembly 200 may contain slits (or slots) 206 in a gel support underneath the gel 201. The slits 206 are congruent to the separation path 205 and align to the center of each microwell 203 of the gel assembly 200. In some embodiments, the width of the slits 206 are about 0.1 mm to about 4 mm, or about 0.5 mm to about 4 mm, or about 1 mm to about 4 mm, or about 2 mm to about 3 mm or about 2 mm to about 4 mm. In other embodiments, the gel assembly 200 contains ribs between one or more separation paths that supply support to the gel separation area defined by the sample wells and separation path.

[0073] In some embodiments, the separation of proteins is achieved in about 2 minutes to about 20 minutes, about 4 minutes to about 18 minutes, about 6 minutes to about 16 minutes, about 8 minutes to about 14 minutes, about 10 minutes to about 12 minutes or any time in- between the two aforementioned times. For example, the separation of proteins can be achieved in about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes.

[0074] With reference now to FIG. 3A, a perspective view of another embodiment of a precast gel assembly 300 is shown. A precast gel assembly 300 may have the same, similar, and / or substantially the same features and functionality, as well as be fabricated using the similar and / or substantially the same method as explained above with respect to precast gel assembly 200. It should be understood that parts with similar numbering, i.e., 203 and 303, describe the same or substantially the same features. For example, precast gel assembly 300 may comprise gel 301 disposed on a cassette comprising cathode reservoir 307, anode reservoir 309, sample well 303, slits 306, and separation path 305 (as seen in inset). In some embodiments, gel assembly 300 may comprise comb 320 that may be used to form wells of a specified dimension in the gel and preserve the integrity of the formed wells during packaging and shipping of the gel. The comb can be inserted into the gel layer directly or into molded well structures to create an even gel height and gel surface when the comb is removed just before use. The use of comb 320 with molded well structures prevent gel solution from fillingKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO the molded wells during gel casting and upon comb removal can create wells with a larger volume capacity compared to wells formed by a comb directly in the gel layer where the well height is defined by the thickness of the gel layer. In some embodiments, gel assembly 300 may comprise raised slit walls 312 on the bottom surface of the cassette that provides a defined channel pathway for protein separation along separation path 305. In some embodiments, slit walls 312 may be flush or slightly below the thickness of the gel. In some embodiments slit walls 312 serve as a hard stop to the compression of the gel by the blotting cartridge during the transfer step.

[0075] Now referring to FIG.3B, an embodiment of sample loading device 350 mated with gel assembly 300 is shown. It should be understood that sample loading device 350 may be used with other gel assemblies disclosed in this document. In some embodiments, sample loading device 350 may comprise sample loading device 350 comprising individual sample cup(s) 352 that each contain a hollow fluid pathway 353 for biological samples to pass through and onto upper surface of the gel. In some embodiments, sample loading device 350 may also contain buffer reservoir 357. In some embodiments, sample loading device 350 may be configured such that the one or more sample cups 352 are each aligned with a slit 306 and a sample separation flow path 305 to precisely deliver biological samples. In some embodiments, sample loading device 350 may be pressed onto a gel 301(FIG. 3A) with suitable force that creates a molded and isolated sample well, i.e., sample well 303 (FIG.3A), that does not leak. In some embodiments, sample loading device 350 may be used with a gel that that is supported by a GelBond® PAG film (Lonza). In some embodiments, sample loading device 350 comprises an attachment means 354 to mate or attach to the cassette side walls in order to maintain a prescribed downward force of the cup loading device against the gel, thus creating the sample wells sealed by the gel preventing sample leakage and cross-contamination. In some embodiments, the attachment means 354 may be inserted into attachment slot 355 on sample loading device 350 to hold and stabilize sample loading device 350 to loading device in place. In some embodiments, attachment means 354 may be different shapes or dimensions such as cylindrical pegs, or clips that attach to interior sidewall of sample loading device 350. In some embodiments, sample loading device 350 comprises support leg 356 that create an offset of the bottom edge of the well cups 352 from the bottom of the gel, thus creating a gap for the sample to migrate through during electrophoresis. In this configuration, bottom edge of sample cup 352 does not contact the top slotted surface of the cassette when sample loading device 350 is mated with gel assembly, e.g., 300.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0076] With reference now to FIG. 4, a side view of another embodiment of a precast gel assembly 400 is shown. A precast gel assembly 400 may have the same, similar, and / or substantially the same features and functionality, as well as be fabricated using the similar and / or substantially the same method as explained above with respect to precast gel assembly 200 and 300. It should be understood that parts with similar numbering, i.e., 203, 303, and 403, describe the same or substantially the same features. For example, precast gel assembly 400 may comprise gel 401 disposed on a cassette 410, and sample well 403 formed by removal of comb 420. In some embodiments, gel 401 may be positioned above one porous layer, or between two porous layers 413 to provide additional structural support and improve the resolution of the separated proteins. In some embodiments these porous layer(s) substantially reduce or block protein electromigration and / or diffusion in the xy plane of the gel during protein separation, while permitting electron flow in the z-dimension allowing for later protein transfer to the blotting membrane. In some embodiments, the gel may be supported and covalently bonded to a GelBond®PAG film (Lonza) sheet with slots to permit protein transfer along the z-axis. In some embodiments, the gel may be supported by a membrane or a track- etched membrane material on one or both sides that impede protein migration along the xy- axis, but is electrically permeable along the z-axis. In some cases, the layer(s) porosity is created by slits or slots cut into the layer along the separation path. In some cases, the layer(s) porosity is created by holes with dimensions in the nanometer to micrometer scale, or by entangled polymers, meshes, sponges, sintering and the like, with openings in the nanometer to micrometer size. In some embodiments the bottom layer is comprised of a membrane typically used in dialysis and has a molecular weight cutoff of 1kDa, or 3kDa or 5kDa, or 10kDa. In some embodiments, a top layer is comprised of a membrane typically used in dialysis and has a molecular weight cutoff of >200kDa or >300kDa, or 500kDa, or 1,000 kDa. In some embodiments, a bottom layer may be comprised of gel polymer, i.e., polyacrylamide gel, of a higher percentage than that of the separating gel and that the bottom layer substantially blocks protein migration, but is still permeable to electrons, e.g., 20-40% polyacrylamide. In some embodiments, gel 401 may chemically bond or crosslink with the porous layer and / or the cassette structure. In some embodiments, a crosslink is formed between the gel acrylamide and acrylate, vinyl, acrylamide, methacrylate groups on the surface of the cassette or porous layer. In some embodiments, the porous layer is a film. In some embodiments, the porous layer or film may be from about 0.02 mm to about 4 mm thick, about 0.05 mm to about 0.1 mm, about 0.1 mm to about 0.2 mm, about 0.1 mm to about 0.5 mm, about 0.5 mm to about 3 mm, or about 1 mm to about 4 mm. In some embodiments, the porous film may include but is notKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO limited to polyethylene, polypropylene, cellulose, porous plastic polymers, cellulose / polymer blend, glass fibers, porous ceramics, graphite, aluminum oxide, porous foams, or combinations thereof. In some embodiments, the porous film is a membrane. In some embodiments, the porous film is a track-etched membrane. In some embodiments, the porous film has a protein molecular weight cutoff of less than about 30 kDa, or less than about 20kDa, or less than about 10 kDa, or less than about 5 kDa, or less than about 3 kDa or less than about 1 kDa. In some embodiments, the porous film is comprised of a material that has low protein binding characteristics. In some embodiments the porous film material may be treated or coated to make it hydrophilic.

[0077] As further seen in FIG. 4, a precast gel assembly 400 may be disposed in a tray 450 positioned under the precast gel assembly 400 and includes a buffer reservoir 453 (that may be configured into cathode reservoir 457 and anode reservoir 459) that holds buffer 451. In some embodiments, tray 450 may be part of gel assembly 400. In some embodiments, the buffer 451 may be running buffer during electrophoresis or transfer buffer during blotting. In some embodiments, the buffer level is such that it contacts the bottom of the gel. In some embodiments, tray 450 may comprise a second cathode that acts as a first electrode in the blotting module. In some embodiments, the second cathode may be embedded in the buffer reservoir 453 or configured to engage with the reservoirs by the instrument. In some embodiments, the electrodes may be disposable (e.g., copper) or permanent electrodes (e.g., platinum) that are inserted into the reservoirs through instrument automation. In some embodiments, gel 401 may track through the inner channel 411 such that gel 401 contacts the buffer 451. In this configuration, after the separation of protein on gel 401 along the y-direction, protein bands in the gel 401 may be directly transferred to the transfer membrane along the z- direction.

[0078] With reference now to FIG. 5, a perspective view of one embodiment of a blotting cartridge 500 is shown. Blotting cartridge 500 may comprise a hollow chamber 501 comprising a first opening 502 on a first side 503 of blotting cartridge 500 where transfer membrane 504 is attached and spanning the first opening 502. In some embodiments, transfer membrane 504 may have a length ranging from about 1 cm to about 8 cm, about 1 cm to about 3 cm, about 3 cm to about 5 cm, about 5 cm to about 8. In some embodiments, transfer membrane 504 may have a width ranging from about 2 cm to about 10 cm. In some embodiments, the dimension of transfer membrane 504 may be configured to be the same, similar and / or substantially the same as the first opening 502 of blotting cartridge 500. In some embodiments, transferKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO membrane 504 may comprise a material selected from, but not limited to, polyvinylidene fluoride (PVDF), nylon, and nitrocellulose.

[0079] In some embodiments, blotting cartridge 500 may comprise a smaller, second opening 505 on a second side 506 opposite the first side 503 of blotting cartridge 500. A hollow, substantially cylindrical body 507 is attached to second opening 505 at a first end 508 and comprises passageway 509 that provides a continuous path from second opening 505 to opposing second end 511, which can be attached to a vacuum or positive and negative pressure source. The second end 511 can also be used to attach to an anode that functions as a second electrode in the blotting module and that provides voltage potential between buffer reservoir 453 (FIG. 4) and blotting cartridge 500 for transfer of separated protein from gel to transfer membrane 504. In this configuration, the chamber 501 provides even pressure across transfer membrane 504 to provide a uniform transfer of protein bands from the gel to the transfer membrane 504.

[0080] In some embodiments, the blotting cartridge 500 may comprise a porous polymeric material support layer (not shown) positioned behind the transfer membrane 504 and spanning the first opening 502 to provide additional structural support to transfer membrane 504, prevent warping of transfer membrane 504, and improve protein band uniformity by allowing the membrane to make even contact across the gel surface during protein transfer and allowing reagents to flow uniformly across the transfer membrane 504 during blotting. In some embodiments, the dimension of the porous support material layer may be suitable to span the opening of the blotting cartridge. In some embodiments, the dimension of the porous support layer may be configured to be the same, similar and / or substantially the same as the first opening 502 of blotting cartridge 500. In some embodiments, the porous support material may include but is not limited to, polyethylene, polypropylene, cellulose, porous plastic polymers, cellulose / polymer blend, glass fibers, porous ceramics, graphite, porous foams, or combinations thereof. In some embodiments, porous support material may serve as the anode of the blotting module.

[0081] Now referring to FIG. 6, another embodiment of a blotting cartridge 600 that may be used in the present method and system is shown. The blotting cartridge 600 may have the same, similar and / or substantially the same features and functionality, as well as fabricated using the similar and / or substantially the same method as explained above with respect to blotting cartridge 500. For example, blotting cartridge 600 may have a hollow chamber 601, transferKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO membrane 604, a first opening 602 on first side 603, second opening 605 on second side 606, hollow cylindrical body 607 comprising passageway 609, and ends 608 and 611. In some embodiments, the blotting cartridge 600 may comprise at least one membrane support rib 613 positioned along the bottom opening 602 of blotting cartridge 600 that provides additional structural support for the module and for the transfer membrane 604. The position of said support ribs are such as to not align to slots on the gel cassette if so present.

[0082] Now referring to FIG.7, another embodiment of a blotting cartridge 700 that may be used in the present method and system is shown. The blotting cartridge 700 may have the same, similar, and / or substantially the same features and functionality, as well as fabricated using the similar and / or substantially the same method as explained above with respect to blotting cartridges 500 and 600. For example, the blotting cartridge 700 may have a hollow chamber 701 with openings 702a, 702b, and 702c, at first side 703 of blotting cartridge 700, transfer membrane(s) 704 (a, b, or c) on first side 703, second opening 705 on second side 706, hollow cylindrical body 707 comprising passageway 709, and ends 708 and 711. In some embodiments, the blotting cartridge 700 may comprise more than one opening (i.e., 702a, 702b, or 702c) configured to attach more than one transfer membrane (i.e., 704a, 704b, and 704c) for a parallel and multiplex transfer and analysis of different proteins that require different immunodetection protocols. In this configuration, bottom openings holding transfer membrane 704a, 704b, and 704c contained within the same hollow chamber 701 provide equal pressure across transfer membranes 704a, 704b, and 704c such that transfer of proteins from gel to transfer membrane and reagents from reagent tray to membrane are uniform across the transfer membrane.

[0083] In this concept, the bottom portion of the cartridge is subdivided into three separate compartments, each with a separate membrane and support layer. As shown, the blotting cartridge includes a top cover that includes a pressure port at top, and multiple chambers with dividing walls in between, and membrane segments 704a, 704b, 704c associated with each chamber. A slit is formed between each compartment that can fit over a complementary dividing wall in a reagent reservoir tray used to present antibodies 1, 2, and 3 to the separate supported membrane segments 1, 2 and 3 on the cartridge bottom. The blotting cartridge can be placed in an appropriate reagent reservoir to load the desired reagents.

[0084] Now referring to FIGS.8A and 8B, another embodiment of a blotting cartridge 800 that may be used in the present method and system is shown. The blotting cartridge 800 mayKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO have the same, similar, and / or substantially the same features and functionality, as well as fabricated using the similar and / or substantially the same method as explained above with respect to blotting cartridges 500, 600, and 700. For example, the blotting cartridge 800 may have a hollow chamber 801 with first opening 802 on first side 803 of blotting cartridge 800, second opening 805 on second side 806, transfer membrane 804 disposed on first opening 802, hollow cylindrical body 807 comprising passageway 809, and ends 808 and 811. In some embodiments, the blotting cartridge 800 may comprise an additional cover 813 that is separately attached to first side 803, i.e., the bottom, of blotting cartridge 800 and holds a second membrane 814, support, or pad 815. In some embodiments, cover 813 may comprise tabs 817 that attach cover 813 to blotting cartridge 800. In some embodiments, cover 813 may be attached to the blotting cartridge by other means of attachment including, but not limited to clamps, magnets, screws, latch, or a lock-and-key configuration. In some embodiments, the means of attachment creates an air-tight seal. In this configuration, second membrane 814 is indirectly attached to the bottom of the blotting cartridge 800 when cover 813 is fit over the bottom of the blotting cartridge 800 to hold the two pieces in place. In some embodiments, second membrane 814 may comprise the same material as transfer membrane 404, 504, 604, 704, or 804. In some embodiments 804 may be comprised of a different material than 814, such as polyether sulfone (PES), PVDF, nylon, nitrocellulose. In some embodiments, pad 815 may be a Bio-Rad Trans-Blot Turbo transfer pad. In some embodiments, when the top and the bottom pieces of the blotting cartridge 800 are pressed fully together, the resulting blotting pressure may provide about 1 to about 2 kg of force to the membrane 814 on the upper surface of the gel.

[0085] Now referring to FIG. 9, a schematic for the transfer of separated protein from a precast gel 955 to a blotting cartridge 900 is disclosed. In some embodiments, the blotting cartridge 900 may be any blotting cartridge in accordance with the present disclosure, including blotting cartridges 500, 600, 700, and 800. It should be understood that parts with similar numbering, i.e., 501, 601, 701, and 801 describe the same or substantially the same features of the blotting cartridge 900. As seen in FIG.9, the precast gel 955 with separated protein bands is disposed in blotting tray 951 comprising transfer buffer 915 and cathode plate 953. In some embodiments, transfer buffer 915 is added to chamber 901 of cartridge 900 and held within chamber 901 by transfer membrane 904. In some embodiments, transfer buffer 915 may comprise a mixture of Tris(Hydroxymethyl)aminomethane and glycine including but not limited to Towbin buffer, Timmons buffer, and Bjerrum Shafer-Nielsen buffer. In someKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO embodiments the transfer buffer is comprised of the Trans-Blot® Turbo™ (TBT) transfer buffer from Bio-Rad Laboratories which is formulated for rapid protein transfers. Further details regarding transfer buffer systems are illustrated and described, for example, in U.S. Patent No.9,150,611 to Chmiel et al., the entire contents of which are incorporated herein by reference. In some embodiments, about 5 ml to about 100 ml, about 5 ml to about 90 ml, about 10 ml to about 80 ml, about 20 ml to about 70 ml, about 30 ml to about 60 ml, about 40 ml to about 50 ml, or about 5 ml, about 10 ml, about 15 ml, about 20 ml, about 25 ml, about 30 ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml, about 60 ml, about 65 ml, about 70 ml, about 75 ml, about 80 ml, about 85 ml, about 90 ml, about 95 ml, or about 100 ml of transfer buffer may be added.

[0086] The blotting cartridge 900 is then lowered into blotting tray 951 and contacts the upper surface of the gel where separated proteins are present. In some embodiments, the blotting cartridge 900 may contact the upper surface of the precast gel at a pressure from about 0.5 kg-force to about 5 kg-force, about 0.5 kg-force to about 1 kg-force, about 1 kg-force to about 1.5 kg-force, 1.5 kg-force to about 2 kg-force, 2 kg-force to about 2.5 kg-force, 2.5 kg- force to about 3 kg-force, 3 kg-force to about 3.5 kg-force, 3.5 kg-force to about 4 kg-force, 4 kg-force to about 4.5 kg-force, to about 4.5 kg-force to about 5 kg-force. Once in contact, an electrical current is applied between cathode plate 953 and anode 913, driving the transfer of protein bands from the precast gel 955 to transfer membrane 904 in a z-direction. In some embodiments, blotting cartridge 900 may be mounted on an XYZ gantry or a movable robotic arm that automatically moves the blotting cartridge 900 between different modules, including the separation module, blotting module, and imaging module. In some embodiments, the transfer of protein bands from the precast gel assembly 955 to transfer membrane 904 is achieved in about 2 minutes to about 10 minutes, about 4 minutes to about 8 minutes, or about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, or about 10 minutes, or any range between the two aforementioned points.

[0087] After the transfer of separated proteins, the remaining transfer buffer 915 in chamber 901 is either aspirated or pushed from blotting cartridge 900 by a vacuum or pressure system (not shown) connected to blotting cartridge 900 at upper end 911 of cylindrical body 907. In some embodiments, the vacuum system is connected to a waste container where excess solutions aspirated from blotting cartridge 900 are stored. In some embodiments, the vacuum system is reversed to apply air pressure to upper end 911 of cylindrical body 907, which pushesKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO the solution though the membrane and back into the tray for disposal, thus the used reagent is returned to the original compartment which is then used as a container for the solution waste. In other embodiments, various other means can be used to move the solutions using positive or negative pressure, for example, a syringe pump, diaphragm pump, parastaltic pump or similar. The transfer membrane 904, which now holds transferred proteins, is optionally rinsed at a wash station positioned within the system. Application of vacuum to opening on upper end 911 of cylindrical body 907 while cartridge 900 is positioned in a reservoir containing water or buffer results in the washing of the membrane. As indicated for transfer buffer, the wash solution can be aspirated to a waste container using vacuum or is deposited into an unused or previously used reservoir by applying air pressure to opening on upper end 911 of cylindrical body 907. In some embodiments this rinsing step is repeated multiple times. In other embodiments, vacuum and air are alternated during the washing to flow the wash solution back and forth across the membrane. In still other embodiments, the cartridge is shaken or agitated while being dipped in the wash solution for more effective washing of the outside surface of the cartridge; the shaking can be a separate step, or steps can be performed concurrently with the application of vacuum or air to upper end 911 of cylindrical body 907.

[0088] Now referring to FIG. 10, a schematic for another embodiment of the transfer of separated protein bands from a precast gel assembly 1500 to a blotting cartridge 1000 is disclosed. In some embodiments, the precast gel assembly 1500 may comprise the same, or substantially the same, similar, and / or substantially the same features and functionality, as well as be fabricated using the similar and / or substantially the same method as explained above with respect to FIG. 9 or any of the precast gel assembly disclosed in this document. In some embodiments, blotting cartridge 1000 may be any blotting cartridge in accordance with the present disclosure, including blotting cartridges 500, 600, 700, 800, and 900. It should be understood that parts with similar numbering, i.e., 501, 601, 701, 801, and 901 describe the same or substantially the same features as described in blotting cartridge 1000. As seen in FIG. 10, the precast gel assembly 1500 with separated proteins is disposed in blotting tray 1051 comprising transfer buffer and cathode plate 1053. In some embodiments, transfer buffer may be added to chamber 1001 of cartridge 1000 and held within chamber 1001 by transfer membrane 1004. In some embodiments, transfer buffer may include same or substantially the same transfer buffer as disclosed in FIG.9 or in this document. In some embodiments, anode 1013, that is used for protein band transfer may be inserted into passageway 1009 of end 1011. In some embodiments, transfer buffer may be vacuum aspirated into blotting cartridge 1000KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO from blotting tray 1051. In some embodiments, precast gel assembly 1500 may comprise gel 1501 disposed on cassette 1510 comprising cathode reservoir 1507, anode reservoir 1509, and sample well 1503. It should be understood that the embodiment of transfer of separated protein as disclosed in FIG. 10 operates in the same, or substantially the same, similar method and protocol, as explained above with respect to FIG. 9 and any other methods presented in this document.

[0089] As seen in FIG.11A, after transfer of the protein bands from gel assembly to transfer membrane of the blotting cartridge, the blotting cartridge 1110 is moved to a reagent tray 1100 comprising compartments 1105 pre-filled with blotting reagents to label transferred proteins for immunodetection. In some embodiments, blotting reagents may comprise blocking buffer, wash buffer, primary antibody solution, primary antibody diluent, secondary antibody solution, secondary antibody diluent, diluent, protein-conjugated reporter molecule, oligonucleotide- conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution. The blotting cartridge can be moved by any suitable means, such as an XYZ gantry 1101a or robotic arm 1101b, as shown in upper right inset.

[0090] In some embodiments, each compartment is pre-filled with about 1 ml to about 10 ml of the blotting reagents. For example, each compartment can be pre-filled with about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, or any amount in-between of the blotting reagents. In some embodiments, the reagent tray 1100 may comprise 5 to 14 compartments 1105 but can have any number required to perform the individual steps of the blotting procedure. In some embodiments, the dimension of compartment 1105 is configured to the dimension of the membrane of cartridge 1110 such that the cartridge may be entirely disposed in compartment 1105. In some embodiments, one or more compartments 1105 may be empty for the end user to provide the desired blotting reagent (i.e., specific antibody or chemiluminescent substrate) to reagent tray 1100. In some embodiments, some empty compartments can contain a diluent solution for the end user to add their reagent of choice; for example, the compartment may contain an antibody diluent and the customer adds their own primary and / or secondary antibody to the diluent completing the reagent. In some embodiments, cartridge 1110 is rinsed and aspirated to remove excess blotting reagent from the transfer membrane between engaging with each compartment 1105. As described above, vacuum is applied to second end 1111 of cylindrical body 1107 of cartridge 1110 to draw the blotting reagents across and through the transfer membrane according to the prescribed protocol and required rate in order to allow sufficient time for blocking, antibodyKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO binding, detection probe binding and the like. Also as described above, vacuum or air pressure can be used to empty the used blotting reagents from the cartridge by either further aspirating them to a waste container or returning them to their compartment in the tray. In some instances, the cartridge can be shaken or agitated during the period before or during the aspiration of the blotting reagents. In still other embodiments, the blotting reagents can be repetitively aspirated and dispensed by applying vacuum and air pressure, respectively, to afford efficient contact with the transfer membrane. In still further embodiments the reagents in the reagent tray can be warmed to accelerate binding reactions and improve background removal. The warming can be at temperatures from about room temperature to about 40C so as not to damage or denature proteins in the reagents. Warming can be performed using any method known in the art such as thermoelectric, resistive, conductive, infrared, heated air circulation types of heating, or any other. As used herein, “air pressure” may refer to positive or negative pressure and can be delivered by any suitable means, such as a pump. As used herein, “vacuum” in the context of the devices and methods desribed herein can include a low pressure vacuum (e.g.100 mTorr).

[0091] As shown in FIG. 11B, the differing compartments 1105 of the reagent tray can be filled with various differing fluids or solutions, including any of blockers, primary / secondary antibodies, wash fluids, and chemi susbstrate. The blotting cartridges can be moved between the differing compartments and a respective fluid can be drawn into the blotting cartridge and / or expelled from the blotting cartridge through pressurized flow, such through a vacuum / pressurization means attached to the port of the blotting cartridge. The blotting cartridge could be moved by an x-y-z robot, or any suitable positioning means. In this embodiment, the reagent tray is disposable. While certain dimensions as shown in FIG.11B, it is understood these dimensions are exemplary and various other dimensions could be realized.

[0092] In one such procedure, the cartridge first moves to engage with the top surface of the gel cassette allowing for protein transfer, then moves to the reagent tray to sequentially walk through the preprogrammed blot processing protocol steps and finally moves to the imaging station to visualize the detected bands on the probed membrane. Other components of the instrument include a vacuum / pressure source, LEDs (e.g., red, green, blue, near infra-red, far infra-red) for fluorescent blotting applications, camera and / or optional contact image sensor, a touch screen, advanced AI-driven Software for automated data analysis and reporting, and a waste container on the outside of the device. The device may optionally contain a UV LED andKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO separate camera positioned above the gel cassette for performing the Stain-FreeTM application for sample normalization.

[0093] The compartments of the reagent tray are typically in 2 x 5 matrix. However, it is appreciated that the reagent tray can be arranged in different formats for example, have less than 10 compartments, be arranged in linear format or in a disc shape. The cartridge can be moved via an XYZ gantry or robotic arm (such as those shown in FIG. 11A) to each compartment or the reagent tray can be translated or rotated to bring the compartment to the membrane cartridge. In some embodiments, the tray (and outside of membrane cartridge) may be made hydrophobic by coating or surface modification in order to reduce any meniscus which can push more reagent than required into the inside of the cartridge. Controlling the height of the cartridge within the solution level during withdrawal and infusion steps can also be utilized.

[0094] The reagent tray and systems described herein can be utilized for performing multiplex western blots. This is typically performed using primary antibodies from different species to different targets and then using anti-species secondary antibody conjugated with different color fluor. In some embodiments, the primary or secondary reporter reagent may be a fluorescent nanoparticle such as a polymer dot or quantum dot. In some instances, the primary or secondary reporter reagent may be a StarBright dye from Bio-Rad. Rapid multiplex western blotting using oligo-labeled antibodies may be performed using the systems or components described herein. Rapid multiplex western blotting can be further understood by referring to U.S. Patent Application Publication No. US2020 / 0209229, which is incorporated herein by reference in its entirety for all purposes. This approach could be adopted for this device. It is appreciated that various reagents can go into the reagent tray, including a release oligonucleotide. The release oligo facilitates the sequential multiplex western.

[0095] In another aspect, the system can be configured to reduce bubbles being trapped between the gel and membrane during the blotting procedure. During the blot processing, bubbles on top of the gel card can be removed by first having the membrane cartridge dispense some of the transfer buffer through the membrane and on top of the gel card creating a small pool of liquid. The cartridge is then lowered which pushes out any remaining bubbles. It is appreciated that other means to remove bubbles such as a puff of air can also be used.

[0096] In yet another aspect, the cartridge and disposable reagent tray can be part of a kit. In some embodiments, the blotting cartridge with membrane may be delivered to the customer inKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO a prewet format containing the required volume of transfer buffer in a disposable tray that can optionally be loaded directly into the instrument during the manual steps of the procedure.

[0097] After contacting and incubating the transfer membrane with the appropriate blotting reagents, blotting cartridge 1110 is moved to the imaging module for colorimetric, chemiluminescent, or fluorescent detection of the transferred protein bands. Now referring to FIG.12A, an embodiment of an imaging module 1200 that may be used with present systems and methods is shown. The imaging module 1200 may comprise an image sensor 1201. In some embodiments, image sensor 1201 may be a camera or CCD. In some embodiments, the imaging module 1200 may comprise an illumination / excitation source 1203 that is configured to emit light in the ultraviolet (from about 100 nm to about 325 nm), visible (from about 325 nm to about 700 nm), or near-infrared (NIR) to infrared (IR) (from about 700 nm to about 1000 nm) range. In some embodiments, the excitation source 1203 may include one or an array of light-emitting diodes (LED) 1205 emitting light at a wavelength ranging from about 100 nm to about 1000 nm. In some embodiments, excitation source may comprise monochromatic laser emitting single wavelength between about 100 nm and about 1000 nm, between about 100 nm and about 200 nm, between about 200 nm and about 300 nm, between about 300 nm and about 400 nm, between about 400 nm and about 500 nm, between about 500 nm and about 600 nm, between about 600 nm and about 700 nm, between about 700 nm and about 800 nm, between about 800 nm and about 900 nm, between about 900 nm and about 1000 nm or may be any other or intermediate wavelength and / or range of wavelengths.

[0098] In some embodiments, cartridge 1210 is positioned above the sensor or camera and light from excitation source 1203 excites labels on the proteins bound to transfer membrane 1211. The resulting signal is detected and collected by camera or image sensor 1201. The imaging module may comprise one or more optical filter 1209 between the image sensor 1201 and transfer membrane 1211 that corresponds to wavelength of excitation source 1203 and corresponding emission wavelength from the label, i.e., fluorescence imager mode. In some embodiments, for example when using chemiluminescent detection, cartridge 1210 may be lowered to directly contact the surface of image sensor 1201, the resulting signal is detected and collected by image sensor 1201. In this configuration, transfer membrane 1204 may be in direct contact with the image sensor 1201, i.e., contact imager mode as seen FIG.12B. Further details regarding the contact imager mode are illustrated and described, for example, in U.S. Patent No 9,794,454 to Uri et al., the entire contents of which are incorporated herein by reference.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0099] In some embodiments, imaging module may be configured to detect and collect colorimetric, chemiluminescent, fluorescent, or Stain-FreeTMoptical data. After collecting the relevant immunodetection signal, the system automatically processes the data, i.e., removes noise, normalizes and ratios its signal to a standard or the Stain-FreeTMsignals collected earlier, identifies the protein molecular weight based on the protein standards, the protein amount and or amount relative to the total signal, and reports these results to the end user in tabular and / or graphical formats. In some embodiments, the process of automated separation, transfer, detection, and analysis of proteins in a biological sample is completed in less than about 5 hours. In some embodiments, the process may occur in less than about 4.5 hours, less than about 4 hours, less than about 3.5 hours, less than about 3 hours, less than about 2.5 hours, less than about 2 hours, less than about 1.5 hours, about 1 hour, or any amount of time in-between. In some embodiments, after imaging and / or detection of proteins, a cleaning pad may be snapped on, or press-fit over surface of the transfer membrane 1211 of a blotting cartridge 1210 and used to clean and dry the image sensor 1201 or camera lens by moving the cartridge / cleaning pad unit back and forth over the sensor or camera lens surface. The cleaning pad is disposed along with the blotting cartridge.

[0100] Another aspect of the present disclosure provides a process for analyzing biological samples using the present system. In some embodiments, the presently disclosed method may encompass different types of protein blotting techniques, such as Western blotting, Eastern blotting, Far Eastern blotting, Northwestern blotting, or Southwestern blotting. In some embodiments, the method may comprise: (a) inserting the gel loaded with the biological sample, molecular weight standard, and running buffer into a blotting reservoir; (b) engaging a separation anode with a separation cathode in the separation module, and applying an electric current between the separation anode and cathode to separate the proteins into a plurality of protein bands; (c) dispensing transfer buffer to the blotting tray reservoir under the gel and into the blotting cartridge; (d) lowering the blotting cartridge toward the upper surface of the gel until the transfer membrane contacts the upper surface of the gel, engaging a transfer anode with a transfer cathode, and applying an electric current between the transfer anode and the transfer cathode to transfer the plurality of protein bands from the gel to the transfer membrane; (e) removing the transfer buffer from the blotting cartridge and optionally washing the blotting cartridge with water or a wash buffer; (f) processing the transfer membrane by sequentially aspirating one or more blotting reagents from one or more individual compartments of the reagent tray through the transfer membrane of the blotting cartridge to form a plurality ofKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO labeled protein bands on the transfer membrane; (g) imaging the labeled proteins; and (h) analyzing and detecting the labeled proteins. In some embodiments, the gel may be loaded into the present system along with the biological samples. In some embodiments, the biological samples may be preheated, or inserted into a heating block inside the device first and the system would dispense the running buffer and samples into the microwells reducing potential user errors, since samples would not be manually loaded into small wells of the gel. In some embodiments, the separation, transfer, and analysis steps are automated within the disclosed system.

[0101] In some embodiments, post translation modifications (PTMs) may be applied before detection and analysis steps, i.e., in an Eastern blot workflow. In some embodiments, specific antibodies or binders to the modification or PTM may be used for detection; for example, antibodies against phosphorylation sites, or lectins which bind to carbohydrate moieties decorating the protein of interest. In some embodiments, PTM binders may be conjugated with a detectable label (fluor, HRP, AP, biotin, etc) or detected using a secondary labeled binding reagent analogous to a labeled secondary antibody. For certain embodiments, the system may comprise a reagent tray having sections that contain one or more PTM binders that contain a detectable label or can be detected by a secondary reagent in the tray that recognizes the PTM binder.

[0102] In some embodiments, instead of binding to an antibody, target protein may bind to a particular DNA or RNA, as performed in Southwestern and Northwestern blotting techniques. In some embodiments, these DNA or RNA oligonucleotides may carry a label or tag that can be detected directly (fluorescent molecule) or indirectly e.g. digoxigenin using an anti-DIG antibody or biotin with a labeled streptavidin. Thus, by adjusting the steps used to aspirate the solutions in the reagent tray, various protein blotting techniques may be performed using the system of the present disclosure.

[0103] Now referring to FIG. 13, a flow chart illustrating an exemplary process 1300 for analyzing biological samples is shown. Process 1300 begins with block 1301, where consumables are loaded into the instrument. In some embodiments, before block 1301, a user may open packaging, remove covers on the top and / or bottom of the gel before use, remove a cover or foil from the plate of reagents before loading the consumables in the instrument. In some embodiments, the transfer membrane may be automatically attached to the bottom opening of the chamber in the blotting cartridge at this step. In some embodiments, the transferKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO membrane may be indirectly attached to the bottom opening of the blotting cartridge by attaching to another piece that fits over the opening. The process then continues to block 1302, where the gel is loaded with a biological sample, molecular weight standard, and running buffer into the gel reservoirs. In some embodiments that do not have integrated reservoir(s), the gel may be loaded with biological samples and then running buffer added to the reservoirs. In some embodiments the gel may first be loaded into the instrument and then the buffer added to the reservoirs and samples and molecular weight standards loaded into the wells. In some embodiments, the biological samples may be mixed with sample buffer and heated to denature the protein before loading onto the gel. In some embodiments, the system may comprise a heating block to automatically heat the samples.

[0104] At block 1303, proteins in the biological sample are separated into protein bands by applying an electric current between the anode and the cathode. In some embodiments, block 1303 proceeds by engaging a separation anode with a separation cathode in the separation module and applying an electric current between the separation anode and cathode to separate the proteins into a plurality of bands. In some embodiments, after the separation of proteins, Stain-FreeTMimaging of the gel by exposing the separated protein bands to ultraviolet light may be performed in block 1304 before the transfer of the separated proteins. At block 1305, transfer buffer may be dispensed into the blotting reservoir and in the chamber of the blotting cartridge. In some embodiments, transfer buffer may be aspirated through the membrane until suitably filled with transfer buffer. In some embodiments, transfer buffer in the chamber may be dispensed to a small degree to remove any bubbles from near the membrane surface. In some embodiments, transfer buffer may be filled to the appropriate reservoir and blotting cartridge at step 1301. At block 1306, the blotting cartridge is lowered toward the upper surface of the gel until the transfer membrane contacts the first surface of the gel, engaging a transfer anode with a transfer cathode, and applying an electric current between the transfer anode and the transfer cathode to transfer the plurality of protein bands from the gel to the transfer membrane. In some embodiments, the transfer membrane may be directly or indirectly attached to the bottom opening of the blotting cartridge. In some embodiments the membrane of the blotting cartridge is pressed against the first surface of the gel at a prescribed force. After transferring the protein bands, the process continues to block 1307, where the transfer buffer is removed from the chamber of the blotting cartridge and washing of the blotting cartridge is performed. At block 1308, the transfer membrane is processed by sequentially aspirating one or more blotting reagents from one or more individual compartments of the reagent trayKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO through the transfer membrane of the blotting cartridge to form a plurality of labeled protein bands on the transfer membrane. As shown in block 1309, the blotting cartridge may optionally be rinsed with water or wash buffer between each aspiration step. The labeled protein bands on the blotting cartridge transfer membrane are then moved to the imaging module, and the optical signals, i.e., colorimetric, chemiluminescence, and fluorescence, are detected in block 1310. After the detection of optical signals, the data may be processed further by removing noise and normalizing the data to a standard and reported to the end user in tabular and / or graphical formats in block 1311. In some embodiments, after block 1310, the membrane is stripped with a stripping buffer and block 1308 repeated using a different tray of blotting reagents to probe for additional target proteins. In some embodiments, a previously used blotting cartridge with bound and stripped proteins on the membrane as well as a reagent tray are loaded into the instrument at block 1301 and the process continued at block 1308 without performing the intervening steps. Sample Preparation

[0105] Sample preparation for use in the above-described system depends in part on the target proteins of interest. In some embodiments, the sample is a biological sample. Biological samples may be obtained from viruses, or any biological organism, e.g., an animal, plant, fungus, bacterial, archaea, protozoan, or any other organism. In some embodiments, the biological sample is from an animal, a mammal (e.g., a human or a non-human primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g., chicken), or a fish. A biological sample may be any tissue or bodily fluid obtained from the biological organism, e.g., blood, a blood fraction, or a blood product (e.g., serum, plasma, platelets, red blood cells; and the like), sputum or saliva, tissue (e.g., kidney; lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue); cultured cells, e.g., primary cultures, explants, transformed cells, stem cells, stool, or urine. In one embodiment, cells (human or animal cells) are lysed with a lysis buffer as known in the art (e.g., RIPA). In one embodiment, samples are prepared from tissues (human or animal tissues) and homogenized or lysed. The protein concentration of a sample can be quantified using a Lowry assay, a Bradford assay, a BCA assay, or UV spectroscopy. The samples can then be prepared using appropriate amounts of sample buffer that may or may not contain a reducing agent. In some embodiments, the reducing agent is β- mercaptoethanol or dithiothreitol (DTT), or tris(2-carboxyethyl)phosphine (TCEP).KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO Buffers A. Running Buffer

[0106] Various running buffers can be used in the above-described system as known in the art. Exemplary running buffers include, but are not limited to: 1. a tris-glycine-SDS running buffer that comprises 25 mM Tris Base, 192 mM Glycine, 0.1% SDS, pH 8.3; and 2. a tricine SDS running buffer that comprises 100 mM Tris Base, 100 mM Tricine, 0.1% SDS, pH 8.3. In some embodiments, the present system may be used to perform non-denaturing gel electrophoresis for investigating protein complexes or native proteins, and to perform blotting of those proteins. In some embodiments these systems may include, but are not limited to protein complexes and DNA protein complexes, or RNA protein complexes. In some embodiments, running buffers for said complexes may include, but not limited to Tris-glycine buffer TG (i,e., no SDS), Tris borate buffer Tris borate EDTA buffer, tris acetate, or Tris acetate EDTA buffer. In some embodiments, additional buffers may include Tris-Tricine-SDS buffer (100 mM Tris, 100 mM Tricine, 0.1% SDS, pH 8.3). In some embodiments, gel electrophoresis can be performed using a cationic detergent like cetyltrimethylammonium bromide (CTAB) in place of SDS in order to improve molecular weight determination of certain hydrophobic membrane proteins or highly charged proteins (e.g. Eley, M. et. al., 1979, Anal Biochem 92(2): 411-419). In these embodiments, the anode and cathode would be reversed to affect protein migration along the separation path and for protein transfer. B. Transfer Buffer

[0107] Different transfer buffers can be used in the above-described system as known in the art. Exemplary transfer buffers include, but are not limited to Towbin buffer (i.e., 25 mM Tris, 192 mM glycine, 20% (v / v) methanol pH 8.3) or Bio-Rad Laboratories’ Transblot Turbo transfer buffer. Additional transfer buffers may include, but not limited to Timmons (i.e., 250 mM Tris, 192 mM Glycine, pH 8.9), 300 mM Tris / Gly+M+S, (i.e., 300 mM Tris, 300 mM Glycine, 20% MeOH, 0.05% SDS pH 9.0) 300 mM Tris / Gly (i.e., 300mM Tris, 300 mM Glycine pH 9.0), Tim+SDS+MeOH (i.e., 250 mM Tris, 192 mM Glycine, 20% MeOH, 0.05% SDS pH 8.9), Towbin+SDS+MeOH (i.e., 25mM Tris, 192 mM Glycine, 20% MeOH, 0.05% SDS pH 8.3). Other transfer buffers include Dunn carbonate buffer (10 mM NaHCO3, 3 mM Na2CO3, pH 9.9); 10 mM CAPS, pH 11; and 10 mM CHES, pH 9.6. It is understood that the type of transfer buffer selected depends upon the pI of the proteins of interest. For example, acidic proteins are generally transferred more efficiently in a transfer buffer with a lower pH.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO Additional transfer buffers may contain a cationic detergent such as CTAB in place of SDS. In these cases, the electrodes in the blotting cartridge and blotting tray are reversed (i.e., the polarity is reversed) to affect the transfer of proteins from the gel to the membrane. C. Wash Buffer

[0108] Wash buffers are used in the reagent trays of the above-described system. Examples of wash buffers include, but are not limited to: 1. 25 mM Tris, 0.15 NaCl, pH 7.2; and 2. 10 mM sodium phosphate, 0.15 NaCl, pH 7.5. In some embodiments, detergents such as Tween- 20 are added to the wash buffers to help remove nonspecifically bound material. D. Blocking Buffer

[0109] Blocking buffers are used in the reagent trays of the above-described system before adding antibodies to probe the membrane for target proteins. Various blocking buffers can be used as known in the art. Examples of blocking buffers include, but are not limited to Tris- buffered saline (TBS) or phosphate-buffered saline (PBS), with or without detergent, wherein the blocking agent is 2-5% non-fat milk, 1-5% bovine serum albumin (BSA), or purified proteins (such as casein), fish serum, gelatin, glycoprotein, or synthetic and / or polymeric blockers like PVA or ficol to name a few. Binding Agent

[0110] The term “binding agent” refers to an agent that specifically binds to a molecule such as an analyte. As used herein, the term “specifically bind” refers to a molecule (e.g., binding agent such as an antibody or antibody fragment) that binds to a target with at least 2-fold greater affinity than non-target compounds, e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 25-fold, 50-fold, 100-fold, or 1000-fold or more greater affinity. While antibodies are described in many contexts herein, it will be understood that other binding agents can be used instead of antibodies as preferred by the user. A wide variety of binding agents are known in the art, including antibodies, aptamers, Somamers (slow off-rate modified aptamers), affimers, lipocalins (e.g., anticalins), thioredoxin A, bilin-binding protein, or proteins containing an ankyrin repeat, the Z domain of staphylococcal protein A, or a fibronectin type III domain. Other binding agents include, but are not limited to, biotin / streptavidin, chelating agents, chromatography resins, affinity tags, or functionalized beads, nanoparticles and magnetic particles.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO Antibodies

[0111] Various antibodies can be used in the reagent trays of the above-described system depending upon the target proteins of interest. As used herein, the term “antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene, or fragments thereof, that specifically bind and recognize an antigen, e.g., a particular analyte. Typically, the “variable region” contains the antigen-binding region of the antibody (or its functional equivalent) and is most critical in specificity and affinity of binding See Paul, Fundamental Immunology (2003). Antibodies include for example chimeric, human, humanized antibodies, or single-chain antibodies.

[0112] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kD) and one “heavy” chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0113] Antibodies can exist as intact immunoglobulins or as any of a number of well- characterized fragments that include specific antigen-binding activity. Such fragments can be produced by digestion with various peptidases. Pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)′2, a dimer of Fab which itself is a light chain joined to VH-C H1 by a disulfide bond. The F(ab)′2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)′2 dimer into a Fab′ monomer. The Fab′ monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0114] In some embodiments, the antibodies are contained in an antibody solution. The antibody solution can comprise 1X TBST or 1X PBS, 1% non-fat milk or 3-5% BSA, and the desired concentration of the antibody. In further embodiments, the antibody solution comprisesKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO sodium azide or other preservative or other protein stabilizer. In one embodiment, approximately 1 mL to 10 mL of antibody solution is used in the reagent trays of the above- described system such as 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, or any amount in-between. In some embodiments, volumes of antibody solution of up to 10 ml may be used.

[0115] In some embodiments, primary and secondary antibodies are used in the reagent trays of the above-described system. In some embodiments, the secondary antibodies are labeled with a fluorescent protein or fluorescent dye, horseradish peroxidase (HRP), biotin, or alkaline phosphatase (AP) or other enzyme reporter molecule. In some embodiments the primary antibodies are labeled with a fluorescent dye or other reporter molecule. In some cases, the primary and / or secondary antibodies are labeled with an oligonucleotide and a reporter molecule is attached to a complementary oligonucleotide directly or indirectly via a nucleic acid scaffold. In some instances, the primary or secondary reporter reagent may be a fluorescent nanoparticle such as a polymer dot or quantum dot. Imaging Analysis

[0116] Various imaging analysis methods can be used in the above-described system depending on the target protein, signal, or immunodetection method of interest including but not limited to colorimeric, fluorescence, chemiluminescence, Stain-FreeTMoptical imaging. In some embodiments, the separated protein bands may be analyzed using an artificial intelligence (AI) software / protocol to characterize the emission profile such as, but not limited to emission wavelength, intensity, peak, spectral overlap. In some embodiments, AI may be utilized to remove noise and / or background signal from the gel and blot images, normalize the signal to standard, calculate quantum yield, and identify the separated protein bands within their designated lanes. In some embodiments the gel and / or gel cassette may contain fiducial marks that assist in defining the gel image orientation, focal plane, focus, identification, standards, gel percentage and type, etc. In some instances, AI may be used to identify the proteins that were detected and report to the user pathway, literature, or automatically make other meaningful biological inferences. In some cases, AI may be used to assess the instrument performance and report when maintenance or service is required. In some cases, AI can be used to aid the user in planning future experiments or suggest appropriate controls to run. Kits

[0117] A related aspect of the present disclosure provides a kit that may be used in the system or method for analyzing proteins in biological samples. The kit may comprise an all-in-oneKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO consumable footprint that may include but is not limited to, a precast gel, a disposable blotting cartridge, a reagent tray, a running buffer solution, a transfer buffer solution, a protein standard, and a cleaning pad. The individual components of the kit may include any one of the embodiments, including, but not limited to, the gel, blotting cartridge, and reagent tray described in this document. In some embodiments, packaging of the kit may contain part, lot number, expiry information, and / or instructions for use on the label. It should be understood any of the components disclosed in FIGS. 1-13, such as gel assembly, cartridge, or reagents may be encompassed by kit 1400. In some embodiments, the kit may include packaging information and instructions for the use of the components in the kit. Referring now to FIG. 14, an exemplary consumable kit that may be used with the present system and method is shown. Kit 1400 may comprise a pre-cast gel 1401, blotting cartridge 1403, transfer buffer 1405, and reagent tray 1407. In some embodiments, the pre-cast gel may be polyacrylamide gel, agarose gel, or a blend thereof. In some embodiments, blotting cartridge 1403 may comprise a transfer membrane (not shown) attached directly or indirectly to the bottom opening of blotting cartridge 1403. In some embodiments, the transfer membrane may be polyvinylidene fluoride (PVDF), nylon, or nitrocellulose. It should be understood that parts with similar components, i.e., blotting cartridges 500, 600, 700, 800, and 900, may be encompassed by kit 1400.

[0118] In some embodiments, the reagent tray 1407 may comprise individual compartments that are pre-filled with various components including but not limited to blocking buffer, wash buffer, primary antibody solution, primary antibody diluent, secondary antibody solution, secondary antibody diluent, diluent, protein-conjugated reporter molecule, oligonucleotide- conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution. In some embodiments, the kit may include a cleaning pad 1411 and a water wash station 1409 for cleaning the blotting cartridge 1403 after engaging with any of the individual compartments in reagent tray 1407.

[0119] In one aspect, one fundamental component of the system is a miniaturized gel cassette. The gel and cassette are designed to be used horizontally instead of vertically as is typical for protein gels. The gel was designed in this fashion to facilitate the automation in moving from protein separation to protein transfer to blot processing and ultimately to imaging. Using a horizontal system effectively uses gravity to assist in forces being evenly applied and solutions remaining level which were envisioned to be important to maintain transfer and immunoblotting uniformity over the area of the membrane. Examples of the cassette and gelKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO card design are shown in FIGS.2A-4 and 15A-15C. The cassette parts are preferably made from ABS-type plastic, although it is appreciated that any suitable material can be used. Appropriate materials may be selected to include attributes such as transparency in the UV wavelength range for performing stain-free protein detection, rigidity to resist deformation from hydrogel swelling forces, ease in applying coatings to resist oxygen permeation or promote adhesion of the gel to the cartridge to name a few. ABS-type plastic is advantageous as it is watertight, rigid relatively stable to moderate heat and compatible with buffers. Typically, the gel card is provided to the user with pre-cast gel. The gel can be cast according to various methods known in the art. The gel and gel card can include any of the techniques or features described in U.S. Patent No. 10,101,296, the entire contents of which are incorporated herein by reference for all purposes.

[0120] FIG. 15A shows a perspective view of a gel card assembly 1500 that includes a gel card 1510 partially inserted into a U-case gel cassette 1520. FIG. 15B shows a cutaway view of the gel card 1510 inside the cassette 1520. As shown, the gel card includes a frame support 1511 that defines a gel layer slot 1522 in which the gel layer is formed, multiple pathway openings 1505 over each separation path for subsequent transfer, and multiple sample wells 1503 along one end to direct the samples into the gel layer. The cassette 1520 includes a top plate 1521, a fill slot 1522, and connector feet 1523 for connecting to the electrophoresis box. In this embodiment, the connector feet 1523 are of a triangular design for interlocking alignment within the box. As can be seen, the cassette has a U-shape design 1524 that connects the fill and gel card slots. Exemplary dimensions for the gel card and cassette components are shown in FIG.15C, although it is appreciated that the dimensions are not so limited and could be varied as needed.

[0121] In some embodiments, the U-cast cassette for the gel card is 3D printed as two parts, a front plate and a back plate with spacers (e.g. ~1.5mm spacers) at the left and right sides to accommodate the thickness of the gel card inserted between the plates. The backplate can include a slot (e.g.1 mm slot) behind where the spacers are located that runs the length of the plate and width of the gel card. In some embodiments, this slot extends 1 mm longer than the length of the gel card. When the top plate 1521 is added, the bottom edge of the slot is closed off and there is only an opening directly under the gel card. Applying tape to the bottom edge of the cassette seals the cassette and makes a cassette cavity that is U-shaped, with one arm of the U containing the gel card, and the other arm an empty slot. In the embodiment shown, pairs of triangular tab connectors 1523 located at the sides of the cassette, and structures moldedKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO onto the back face of the back plate interlock with corresponding features in the base of the electrophoresis box. These features are present to keep the cassette secured in a proper position within the box and resist the force of pushing / pulling on the gel card when extricated from the cassette post electrophoresis. Additionally, these features create an ion flow dam underneath the cassette that impedes ion flow through the buffer on the outside of the cassette. One or more separate buffer dams can be added atop the cartridge before electrophoresis, such as by inserting bars that snap into the holes on the triangular tabs of the top plate, as shown in FIGS. 16A-16B. The use of these buffer dams ensure the current flow between anodes and cathodes of the electrophoresis assembly flows through the resolving gel layer, which provides more uniform separation of proteins in the gel layer, and not above or below the cartridge. As shown in FIG.16A, the ion flow dam 1600 includes a bar or elongated member 1610 and connector portions 1601 at each end that connect with the triangular tabs of the cassette to secure the dam in place. The dams are made of a non-conducting material, such as a polymer or any suitable material. While a specific dam design is shown, it is appreciated that the dam and / or connector could be connected or integrated in various other ways as well. A buffer dam could be directly incorporated into one or both the top and bottom plates during fabrication though later adding a dam to the cassette reduces the cassette form-factor simplifying manufacturing. FIG. 16B shows two dams in place with one dam 1600’ at a proximal position near the row of sample wells and another dam 1600’’ at a distal position nearer the end of the separation path. It is appreciated however that a single dam could be used, either a proximal or distal position or at any other position therebetween. Failure to use these or similar dam structure(s) to impede current flow above / below the gel results in longer electrophoresis times. While two buffers dams are depicted, it is appreciated that a single buffer dam could be used, for example, either of those shown.

[0122] In another aspect, the gel card cassette is designed such that the gel card can be pushed out of the cassette after electrophoresis to facilitate the transfer step and subsequent imaging analysis. In some embodiments, the gel card is pushed out of the gel card by using a blade, but other embodiments may be designed to hold the gel card in place and move the cassette instead or to pull the gel card from the cassette. In some embodiments a blade is used to push the gel card out of the cassette by pushing from the bottom edge and in other embodiments by pushing the top of the gel card such as from the raised edge of the sample well. In some embodiments, the gel card can be pulled out of the cassette by using a connector attached to the well structure, an example of such an embodiment being depicted in FIG. 17. As shown, the gel card 1700,KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO similar to previous embodiments, has a frame 1710 defining a gel slot and openings 1705 above each separation path, and sample wells 1703. However, this embodiment includes an integrated connector 1712 on a proximal end thereof, which can be coupled to a transfer mechanism to pull the gel card out from the cartridge and into a transfer portion of the system. While a particular connector design is shown here, it is appreciated that the connector could be any feature suitable for interfacing with a transport element, including but not limited to a protrusion, hook, recess, or hole. It is appreciated this gel card could be used in a similar cartridge design as in previous embodiments, Further, buffer dams can be included to block ion flow above the gel to ensure more uniform consistent electrophoresis.

[0123] FIGS. 18A-18C show another exemplary gel card design. In this embodiment, the gel card 1810 is integrally formed as a single piece. As in previous embodiments, gel card 1810 includes a frame 1811 having defined therein a gel slit(s) for the gel layer, a row of multiple sample wells 1803 and a series of elongated openings 1805 over the separation paths. In this embodiment, however, the gel layer is separated into separate pathways by use of multiple gel channels 1812, as can be seen in the gel card edge views in FIG.18B and 18C.

[0124] Such gel card embodiments could be 3D printed, injection molded, or formed by any suitable process. In some embodiments, the gel card can be formed of ABS, as described above, or any suitable material. Since the gel card contains small microchannels (e.g., sub- millimeter scale, such as 0.8 mm or less) at the top and bottom (visible from top view), production parts may utilize injection molding of two pieces that are bonded / fused together to recreate the channel. In this embodiment, the gel card contains 12 integrated sample wells (e.g.3x3x5mm, max volume 30ul) at one end. The wells are arranged with a suitable pitch (e.g. 4.5 mm pitch) so that they are compatible with loading using a multichannel pipettor. It is appreciated that various other numbers of wells or pitches could be utilized. These molded sample wells are also more stable, and have a controlled size compared to wells formed from polyacrylamide as seen in standard gels making them easier to use. The wells are seamlessly connected to what are essentially separate gel strips via a long microchannel (e.g. 5mm long) of suitable height (e.g.0.5-1.0 mm, 0.8 mm height). A second 5 mm microchannel section is present at the end of gel strips. In the middle, between the microchannel zones, the gel is exposed through the entire thickness of the gel card (e.g.30 x 2 x 1.5mm). This plus (+) shaped gel cross-section is illustrated in the edge views shown in FIG.18B. Of course, it is appreciated that other shapes and dimensions are possible for the gel strip channel, for example it could be hexagonal, or circular. This middle section of the gel card is where the proteins are separatedKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO and since the gel will be exposed once the gel card is removed this region permits the transfer of the separated proteins to the membrane later in the process. Interspersed between each gel strip is support material, sometimes referred to as “ribs” 1815 that give the gel card rigidity and protect the individual gel strips from damage during card extraction. Likewise, the gel cross- sectional shape helps to lock the gel into the support which preserves the gel integrity during gel card extrication from the cassette.

[0125] In another aspect, the gel card includes a microchannel region connected to the sample wells that is filled with low percentage gel, so it becomes a stacking region. Typically, the stacking gel is extended to the first few mm of the slotted region as well so that banding is offset from the top edge of the slot and all MWs of proteins can be transferred out of the gel without being impeded. The use of the microchannel sections in the design has the added advantage of stopping the flow of acrylamide monomer solution into the sample wells (due to capillary forces) as the gel is cast. This is useful as there may be no need for a gel comb (as is standard practice) saving manufacturing labor and cost.

[0126] In another aspect, gel electrophoresis is performed in a U-cast cassette / gel card that is mounted into a horizontal electrophoresis gel box. Such an embodiment is depicted in FIGS. 19A-19D. It is appreciated this system can be incorporated into an automated system, such as that depicted in FIG. 1 and that any or all of the steps described herein can be performed automatically, for example by an automated robotic arm, and coordinated by a central controller by programmable instructions accessed by the controller.

[0127] FIG.19A shows a top view of electrophoresis and transfer box 1900, which includes an electrophoresis portion 1910 in which electrophoresis is performed, and a ramp 1920 to a transfer portion 1930 where transfer of proteins to a transfer sheet is performed. The gel card inside U-cast cassette 1501 (as shown in FIG.16B) is disposed in the electrophoresis portion. The cassette is aligned and secured by interfacing features, specifically triangular slots 1923 that receive the triangular connectors 1523 of the cassette. The box gel can include electrophoresis electrodes or electrical connectors / wires to power electrophoresis electrodes integrated within the cartridge and / or gel card, a stainless-steel transfer electrode in the transfer portion and TBT pad (yellow) on top of plate electrode.

[0128] FIG. 19B shows a side view with gel card cassette 1501 in gel electrophoresis position where electrophoresis can be performed by the anode and cathode electrodes. It can be seen that the cassette can be slid vertically and secured into position via connectors 1523 inKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO slots 1923. The triangular shape ensures proper alignment within the position above the plate electrodes, although it is appreciated that various other shapes and types of connectors and alignment features could be used. The box further includes a transfer element 1921 that, after electrophoresis is performed, removes the gel card from within the cassette and transfers the gel card into the transfer potion of the box. In this embodiment, the transport element includes a thin blade that is inserted into the gel card slot of the cassette to push the gel card out and then up the ramp and into the transfer portion. In this embodiment, the blade can be advanced (in direction of arrow) into and through the cassette to push the gel card out of the cassette, up the ramp and into the transfer portion. The blade could alternatively extricate the gel card by pushing on the edge of the sample well structure and then move it up the ramp. The system can perform this transfer automatically after electrophoresis is complete. While a blade is depicted here, it is appreciated that the transfer element can be any suitable element that removes the gel card from the cassette (by pushing or pulling) and moves the gel card to the transfer position. In some embodiments, the gel card includes a connector (such as in FIG. 17) that interfaces with the transfer element to facilitate pushing or pulling of the gel card from within the cassette such that the transfer element may be a hook or wire that pulls the feature from the direction of the transfer portion.

[0129] FIG.19C shows a top view of the electrophoresis and transfer box 1900 showing gel card 1510 moved to the transfer portion 1930 and empty U-cast cassette 1520 remains in the electrophoresis portion. The transfer portion includes one or more electrodes to facilitate transfer of the separated proteins from the gel onto a transfer sheet, as described herein. In this embodiment, the transfer portion includes stainless-steel transfer electrode 1931 and a TBT pad 1932 on top of the plate electrode beneath gel card. FIG. 19D shows a side view of gel card 1520 in the transfer position.

[0130] In this particular embodiment, the gel box contains platinum wires in the cathode and anode sections in the electrophoresis portion that engage / activate the cathode and anodes of the gel card cassette assembly. Adjacent to the cathode are ramps that lead up to a second region with a stainless-steel plate electrode. In some embodiments, the ramps are 3D printed as separate parts that are fixed to the sidewalls using double-sided adhesive. This way ramps with different shapes, slopes and curvatures can be explored to optimize the path of the gel card from the cassette to positioning on the transfer electrode, as shown in FIG. 19B. As shown, the box can be integrally formed as a single component. In this embodiment, the gel card is extricated from the cassette after electrophoresis using a blade. The blade may be furtherKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO shaped (for example curved) to promote even force and control its pitch at the top of the ramp. In some embodiment, the back edge of the sample wells has a notch so that the blade engages with the gel card without slipping out of place during gel card translation. Alternatively, the gel card may have one or more features like a knob, handle, hook, etc. on the edge opposite from where the blade pushes so the gel card can be grabbed and pulled from the cassette and up the ramps. In some embodiments, the wall at the top of the ramp has a small cutout so that the gel card and gel do not scrape against the wall as the gel card passes over to the transfer region of the box. To run a gel and position the gel card for the transfer step the user performs the following steps:

[0131] In an exemplary, method the following steps are performed. 1. Prepare the cassette for processing (e.g. remove the tape from the bottom of the cassette exposing the bottom edge of the gel card). 2. Fit the cassette into the gel box into the electrophoresis position (e.g. by inserting the triangular tabs into the corresponding cutouts in the box, and pressing the gel cassette down until the bottom buffer dams engage with the base of the box). 3. Attach one or both of the removable ion flow buffer dams on the top face of the cassette. 4. Add buffer (e.g. Tris-Glycine-SDS (TGS) buffer) into the cathode and anode chambers until the sample wells are covered. Typically, the level should not be above the top of the buffer dams.) 5. Rinse the sample wells (e.g. with TGS buffer using a p1000) to make sure there are no bubbles trapped in the wells. 6. Add Trans-Blot Turbo pad of suitable size (e.g.2.4cm x 5.4cm) onto the stainless-steel electrode if not already present. 7. Wet the pad with transfer buffer (e.g.6ml of 1X Trans-Blot Turbo transfer buffer) 8. Prepare samples (e.g. by mixing with Laemmli sample buffer + reducing agent and heating for 5min at 100C) 9. Load samples and protein molecular weight standards (e.g.5-10 ul or more of sample per lanes 2 to 11, max vol of well typically about 30 ul and 5 ul of Bio-Rad Precision All Blue or Dual Color protein standard to lanes 1 and 12). 10. Activate the cathode and anodes to perform electrophoresis. 11. Perform electrophoresis (e.g. SDS-PAGE at 200V for 20 min) where the proteins are separated (e.g. from 10kda to 250kDa range).KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO 12. Remove gel card from cassette (e.g. by use of a blade pushed firmly, but steadily on the edge of the sample wells to slowly push the gel card out of the cassette or by pulling the gel card from the cassette. 13. Transfer the gel card out of the cassette (e.g. along a ramp). 14. Place the gel card into a transfer position in the transfer portion (e.g. push the gel card until it drops down into a transfer cavity over the prewet Trans-blot Turbo pad and stainless-steel electrode in the transfer portion). 15. Perform protein transfer.

[0132] FIGS. 20A-21B demonstrate the results of the above described method. FIG. 20A. shows the separation of all the blue MW protein standard on U-cast cassette, such as that described above. As can be seen, the separation distance is about 2cm. FIG. 20B shows the ImageJ peak profile of lane 1 showing peaks from 250kDa to 10kDa. FIG.21A shows 5 ul of all blue MW standards separated on a 12% miniProtean TGX gel run until dye front migrated 3 cm. As can be seen, distance from 250kDa and 10kDa MW proteins is about 2cm. FIG.21B shows the ImageJ peak profile of lane 2 showing peaks from 250kDa to 10kDa. To assess the band resolution of the U-cast gel card, protein MW standards were separated and a colormetric image recorded (e.g. using a ChemidocMP), shown in FIG.20A. Gel lane profiles of this image were analyzed using ImageJ as described before (FIG 20B). For comparison, the gel and lane profile of All Blue standards electrophoresed for a similar distance (e.g. ~2cm) on a standard miniProtean TGX gel (e.g.12%) is also shown FIG.21A and 21B. It is evident that the band resolution in the gel card is comparable or better than that normal gel when separated for the same distance. The 15 and 10 kDa peaks are not yet fully resolved on the traditional gel.

[0133] It is appreciated that variations of the above noted gel card design could be realized. As described above, the preferred format for the gel card was a horizontal system to take advantage of gravity and to keep fluids across the membrane more uniform for reproducible western blotting. It also seemed to be more complex to automate a vertical gel electrophoresis and transfer system to a horizontal system for blot processing. Nevertheless, it was also demonstrated that a vertical gel card could give excellent gel separations and the proteins could be effectively transferred to a membrane using an inverted TBT-like setup.

[0134] FIGS.22A-22C shows another exemplary view of a gel card cassette with integrated buffer reservoirs. FIG.22A shows a top view of cassette 2200. The cassette includes a frame 2210 defining an interior gel slot for the gel layer, integrated sample wells 2203 opening to theKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO gel layer, and support ribs / slots 2205 along the multiple separation paths, as well as a cathode 2201 and anode 2202 with integrated buffer reservoirs, the anode region further including trough 2202a. FIG.22B shows a cutaway view through the middle of a separation path lane. In this embodiment, the polyacrylamide gel runs from sample well under the wall of the cathode buffer reservoir and the bar adjacent to it, fills the slots, and under the anode buffer reservoir wall and into the trough at the front of the anode reservoir.

[0135] In another aspect, the gel cassette design might still be viable for development if further improvements in band resolution can be made as the workflow would be familiar to users, and the design affords an easy transition from electrophoresis to protein transfer (e.g. cartridge interfaces with top face of gel and pushes cassette down against a TBT buffer-wet pad positioned on the second transfer electrode beneath the slotted region). In an exemplary cassette, channels are 2.5mm wide, channel walls are vertical and there is 0.4 mm thick gel slab underneath to anchor the gel.

[0136] FIG.22C shows a cutaway view of a cassette 1600 with integrated buffer reservoirs and channel design as described above. As shown, there is a back cavity 1601 (e.g. 0.4mm) and wide channels vertical walls 1602 (e.g. 2.5 mm wide). In some embodiments, the cavity at the back could be eliminated and the channel shape changed to another shape, for example, plus-shaped (+) or hexagonal-shaped (like the gel card) with a top / bottom slot opening (e.g. 1.5 to 2mm) to anchor the gel within the channels themselves. Alternatively, a chemical coating such as an acrylate group could be incorporated onto the cassette surface to crosslink the gel to the support and eliminate issues of gel detachment on storage or use. If the gel does not tear when the top cover is removed, these changes would make the gel thinner overall, improve gel hydration if it is still necessary, possibly reduce electrophoresis time, and improve protein transfer efficiency.

[0137] Previous gel card designs showed twelve sample wells, but the gel card can be made with any number of wells. For example, it could have narrower slots and have three rows of eight samples each in a staggered format to increase the instrument throughput. In this embodiment, the channels connecting the sample wells and the gel slots are different lengths so they could have different cross-sectional dimensions so that the samples from each row arrive at the stacking gel at the same time. In this embodiment, the gel card cassette is about 1.5 times larger to accommodate the additional sample lanes with a pitch of nine mm for loading using a multichannel pipet. FIG.23 shows another exemplary gel card 2300, theKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO design of which allows for a higher number of sample wells. As in similar embodiments, this gel card includes a planar frame 2310 having sampling wells 2303 defined along one end and openings 2305 over each separation pathway. In this design, however, the sample wells are staggered, such that the separation pathways are not limited by the width of the sample well. This allows for a higher density of sample wells and pathways.

[0138] FIGS. 24A-24D shows another gel card design referred to as the S-gel design. This concept is somewhat similar to standard mini slab gel with a first vertical section A comprised of a stacking gel and containing a standard gel comb, a second horizontal middle section B comprised of the slot and ribs design described in other embodiments and separating gel, and a third vertical section C comprised of more separating gel. The electrophoresis box would include three sections, the cathode reservoir 2410, the intermediate separation section 2420 and the anode reservoir 2430, The gel would mount in a reusable box for electrophoresis with the first well section protruding up into a cathode reservoir 2410, and the end of the third section C dipping into an anode reservoir 2410. For casting, a tape would seal the bottom edge, and a film and tape would cover the top and back sides of the slot region, being removed just before electrophoresis. The middle section under the slots would contain the transfer electrode and TBT pad.

[0139] The protocol for electrophoresis and protein transfer with the S-gel design may be as follows. In this embodiment, the cathode box is removable, maybe slides in tracks against the side wall and middle transfer reservoir box. The user removes the films covering the slots, then inserts the gel into the slit of the cathode box through a gasket that seals around the gel, removes the comb and sealing tape on the bottom edge of the cassette. The user then inserts a sponge or pad in a designated area of the middle section of the electrophoresis box and above an electrode such as a stainless-steel plate (e.g. raised feature on bottom to guide placement of sponge / blotting stack / Trans-Blot Turbo pad). The user then slides the cathode box + gel onto the full box frame such that the middle portion of the gel is touching or almost seated against the pad, and the lower portion is dipping inside the anode reservoir. TGS running buffer is added to the cathode and anode boxes to the fill line, and Trans-Blot Turbo transfer buffer is added to the protein transfer sponge to fully saturate. Electrophoresis, followed by protein transfer are automatically performed when the blotting cartridge mates to the top side of the cassette and the under side of the slotted region is pressed against the bottom of the Trans-Blot Turbo pad by pushing the gel down or raising the middle section of the box. It is appreciatedKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO that variations of this design may be realized and still utilize similar concepts as those described above.

[0140] Another aspect of the invention pertains to blotting cartridges, in particular a multipurpose membrane blotting cartridge. This cartridge is a multifunctional device that serves as the bridge that unifies protein transfer, blot processing and blot imaging in the workflow. During protein transfer, the cartridge serves as the upper buffer reservoir (anode) where the proteins are electrophoresed out of the gel and onto the membrane. Afterwards, the cartridge becomes the sealed container required for pressure-driven or electrophoretic-driven immunoblot processing, and finally, it serves as the vehicle transporting the probed blot to the imaging area.

[0141] FIG. 25 shows an exemplary membrane cartridge 2500. It is a rectangular structure sized to fit the top face of the separating gel or gel card that is run horizontally and covers the zone where the gel strips and slots are located. In some embodiments, the base cartridge is manufactured using 3D SLA printing using ABS watershed plastic as described previously. In other embodiments, injection molding techniques may be used which can utilized various different material. The main features of the cartridge are a first open face 2502 on one side, and one or more ports 2511 located on an adjacent or opposite second face. In this embodiment, the openings are on the bottom (first opening) and top (second opening) faces. In other embodiments, the first opening (face where membrane is attached) may be on a side face such that the cartridge can contact and accommodate gel strips that are arranged vertically. On the inside of the cartridge are features 2531, 2532 near the first opening configured to holdup a nested porous support layer. In some embodiments, this layer is formed of porous polyethylene, such as that from Porex Corporation, of suitable thickness (e.g.1 / 8”, 45-90um). An additional rib 2533 that traverses the center of the cartridge adds further support for the porous layer (e.g. Porex layer) in the center. This central rib can be offset slightly compared to the other two support structures so that the porous layer has a slight bow outward in the center which facilitates uniform contact of the membrane against the gel. Other features of the cartridge include connectors behind where the porous layer is mounted to attach platinum wire which serves as the electrode (anode) in this embodiment. In some embodiments, the membrane and support are rigid. In other embodiments, the membrane and support are flexible so that the membrane contacts the gel in the center and then with applied force flattens to complete the full contact, eliminating bubbles in the process. The one or more ports on the second face are used for connecting to a source of positive and negative pressure such as aKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO syringe pump and to an electrode, which is typically located inside the cartridge and behind the porous layer support. It is appreciated that the ports could be in different locations in some embodiments, though the location of the pressure port in the center top face may help promote even pressures and uniform flows across the membrane covering the first opening.

[0142] Before use in the system, the cartridge must be further assembled. This assembly can be performed at the time of use, or the cartridge could be provided partially or fully assembled and provided to the customer or end user. To assemble the cartridge shown in FIG. 25A, a platinum wire is first threaded back and forth through the holes in the attachments formed on the inside to create a wire mesh across the opening 2502 or can be a wire mesh material, as shown in FIG.25B.

[0143] It was found during testing that the position and density of the wire was important to achieving the desired currents to efficiently transfer the proteins from the gel to the membrane. A single wire in the cartridge showed very low currents and required very long protein transfer times, and often in an incomplete fashion. The wire mesh was more efficient for transferring proteins when it was close to the membrane compared to being positioned nearer the top of the cartridge. Thus, the current density near the gel location was an important consideration to achieving fast and complete protein transfers. An electrode plate, such as a titanium-coated metal could substitute for the platinum wire. In some embodiments, the electrode plate includes holes to allow liquids and air to pass for blot processing. Other conductive materials such as graphite sheets could also be used and these may optionally make the electrode a disposable component of the system. In some embodiments, currents of around 0.3A at a voltage of 40V were sufficient to completely transfer protein standards of MW 250kDa to 10kDa in about 7-8 min. In some embodiments, currents of around 0.3 A at a voltage of 40 V were sufficient to completely transfer protein standards of MW 250 kDa to 10 kDa in about 7- 8 min. In some cases, 5400 Joules was used to control for differences in current density between assembled cartridges, and normalize transfer times. Thus, a normalized transfer time in seconds could be calculated as 5400 J / (amps * voltage).

[0144] Once the platinum wire was installed into the cartridge, a porous layer (e.g. ~ 22mm x 54mm piece of Porex X-48971 / 8” thick) was pressed into the first opening and against the support ribs with the smooth side facing out. The porous layer should fit tightly into the opening and typically be flush with the rim of the cartridge. Finally, a membrane was attached to the outside of the cartridge covering the first opening and the Porex layer. The membrane wasKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO attached to the cartridge using a double-sided adhesive applied around the sides adjacent to the opening containing the Porex layer. The membrane could also be applied using an adhesive positioned along the bottom rim of the first opening. Any means of membrane attachment or adhesive that is compatible with the reagents and process could be used.

[0132] Several other variations to the blotting cartridge design have been considered as shown in FIGS.27-29. In some of these variations the cartridge is split into two pieces, a top piece and a bottom piece. In some embodiments, the electrode is part of the instrument. Often the electrode, is a wire (e.g. platinum wire) or plate electrode (e.g. stainless or titanium plate). In the designs shown, the top piece of the cartridge would become a reuseable component associated with the instrument, while the bottom piece which contains the membrane where immunoblotting is performed would be a consumable component. It is appreciated that various differing types and sizes of electrodes could be used. Additionally, splitting the cartridge in this fashion reduces the waste generated from each test making it more environmentally friendly to end users compared to the single piece cartridge.

[0133] FIG. 27 shows embodiment 3200 using a platinum wire 3252 that is separately attached to a frame 3251 that is inserted into the more permanent top piece 3250 containing a pressure port 3211. In this embodiment, the wire has a serpentine configuration where the wire snakes back and forth through holes and a groove at each short end of the rectangular frame creating a continuous wire spaced as an array of parallel wire segments. The end of the wire would exit the cartridge or contact a wire connector to connect it to a power source. This means to connect the electrode to a power source is not shown but is well known in the art. A consumable bottom piece 3260 contains a porous layer 3261 (e.g. Porex) and transfer membrane 3262. In this design concept, the top and bottom portions have a design such that the top piece inserts into the bottom piece creating a sealed compartment capable of performing protein transfer and immunoblotting. The interfacing surfaces of the top and bottom portions can utilize any seal-tight type interface, including those used in the union of a pipettor with a pipet tip. The nesting of the two pieces is such that the wire electrode is positioned close to the back of the porous layer to generate high current density during the protein transfer step. Keeping with the pipettor-tip type interface, the instrument may have a means to push on the edge of the bottom piece and eject the consumable piece away from the top portion.

[0134] FIG.28A shows a fully assembled embodiment of membrane blotting cartridge 2800 with wire electrode 2812. Blotting cartridge 2800 similarly includes interfacing top and bottomKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO portions similar to that described in FIG.27 and can include any of the same or similar features. FIG.28B shows a cutaway view showing the nested permanent and consumable pieces.

[0135] FIGS. 29A-29C shows different views of an embodiment 3400 of a two-piece cartridge using a titanium-coated perforated electrode plate 3452. Otherwise, the embodiment includes same or similar components as the embodiment of FIG. 27 and corresponding numbering. FIG. 29A shows a disassembled view, FIG. 29B shows an assembled view and FIG. 29C shows a cutaway view. The plate electrode would engage a contact or wire that would exit the cartridge to connect it to a power source. This means connecting the electrode to a power source is not shown but is well known in the art. The degree of perforation may control the current density and thus the speed of protein transfer. In some embodiments, the electrode is covered with transfer buffer during the protein transfer process but preferably does not contact the antibody solutions during the blot process steps since it is a reuseable component and could contribute to run-to-run contamination. The risk of this happening should be relatively low as the solution volumes being considered are not large enough to make contact with the electrode. In embodiments where antibody may come in contact with the electrode, for example, when using an electric field to perform blot processing, a disposable electrode, such as one made of graphite, could be used or the top portion of the cartridge could be made easily removable by the user to clean between runs.

[0136] FIG. 30 shows a membrane blotting cartridge 3000 with test fixture 3050. In this embodiment, the blotting cartridge 3000 includes a rigid bottom membrane support 3010 and a top cover 3020. The top cover 3020 includes a pressure port 3021 as well as a separate port 3022 for insertion of an electrode. Top cover 3020 also includes a centrally positioned feature 3023 that interfaces with a test fixture 3050 having a spring-loaded levered shaft to apply downward force on the cartridge to effectuate the required level of contact of the cartridge with the top face of the gel. As can be seen, the pressure port 3021 is offset to make room for the positioning feature. The pressure port can include pressure septa 3021a. Also, in this concept the membrane was bonded to a frame with clips that attached to the cartridge chamber. A gasket material between these pieces created the required seal. To support the membrane, the porous layer was replaced or augmented with a molded piece, a rigid substrate 3010 containing a 2D array of conical features 3015 on its bottom face and the surface is interlaced with through- holes 3014 to allow flow of fluids and air needed for blot processing. The rigid substrate also includes coupling features 3012 for coupling with the top cover 3020. The conical featuresKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO press against the back of the membrane or porous layer (e.g. Porex) for support but minimize the degree of contact to not affect the transfer of proteins but still allow even fluid flow.

[0137] In another aspect, the cartridge may be designed for electroelution. FIG. 31A-31C show an electroelution cartridge, in accordance with some embodiments. FIG. 31A shows a transparent view of an assembled electroelution cartridge 3100, FIG.31B shows an exploded view, and FIG. 31C shows a cut-away view of an assembled electroelution cartridge and the electroelution grid. As shown, the electroelution cartridge 3100 includes the cartridge top 3110 with pressure port / connector 3111, cartridge bottom 3112, electrode 3120, MWCO membrane 3130, gridded compartment layer 3140, low binding / MWCO membrane 3150, support layer 3160, and low binding membrane 3170.

[0138] As indicated previously, the membrane cartridge can be adapted for nucleic acid separation and transfer. It could also be used to perform electroelution of said nucleic acid fragments from the gel card (agarose gel card). A matrixed electroelution grid with a membrane on both sides creates separate buffer-filled chambers where the separated DNA or RNA fragments can be electrically deposited for later recovery by the instrument or the user. Separate columns of chambers align with each lane of the gel card. Membranes could be molecular weight cutoff (MWCO) membranes that permit only certain desired sizes of nucleic acid fragments to pass through. For example, the membrane on the bottom could permit DNA sizes of < / = 1000bp to pass while excluding larger fragments. The membrane on the back side of the grid could block sizes greater than 200bp but permit smaller fragments to pass out of the gridded chambers, thus retaining DNAs with a size range of 200-1000bp. By overlaying the gel image of the size separation, the user can be informed as to which chambers to pool for downstream processing. In other embodiments, 96 samples with sheared nucleic acids could be arrayed in gel card and the desired sizes, for example 200-800bp could be recovered from each chamber in the electroelution device for downstream applications such as DNA library preparation.

[0139] In another aspect, the system can include a filtration station for blot processing with a pad. This allows for high sensitivity blotting in a short time. FIG.32A shows a schematic of such an automated system 3200 that includes a separation unit 3210, a blotting station 3220, an imaging station and a filtration station 3240. The units / stations are coordinated by a central system control and can utilize an x-y-z gantry or any suitable positioning means. It is appreciated that this system can encompass the system shown in FIG. 1. The filtration unitKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO could utilize any suitable filtration means known in the art. The filtration station could utilize a membrane like a PES or cellulose membrane supported by a fluidic network to evenly distribute and filter solutions across the membrane. In some embodiments, during instrument setup, the user would place a pad (e.g. TBT pad of ~ 3 x 6 cm) on top of the filtration stage. During the blot processing process, the membrane cartridge is moved by any suitable positioning means (e.g. XYZ gantry or robotic arm) so that the membrane is in contact with the pad on the stage. Using positive pressure to the cartridge via the port on the second opening, the transfer buffer inside is infused into the TBT pad until the cartridge is empty. The cartridge then moves to a water wash station and water is withdrawn from the reservoir through the membrane and into the cartridge. During this time, the house vacuum is turned on to dry the TBT pad on the stage and remove residual transfer buffer absorbed in the pad; the vacuum is then turned off again. The cartridge containing water wash is mated to the pad on the stage and positive pressure again applied to push the water out of the cartridge and into the pad and ultimately to a waste receptacle. In some embodiments the cartridge is then moved to a buffer reservoir to aspirate the buffer through the cartridge membrane and into the cartridge to equilibrate the membrane containing the transferred proteins prior to the blocking step. The cartridge is again moved to the stage with the house vacuum off and the positive pressure applied to the cartridge to force the water back through the membrane and into the pad on the stage. This kind of cycling of the membrane cartridge between the aspirating western blot solutions from the reagent tray, through the membrane, and then out of the cartridge to the pad on the vacuum stage is repeated many times during the protocol to achieve the blotting result. Controlled aspiration (negative pressure) and positive pressures are used throughout so that antigen and bound antibodies are not removed from the blot during processing. The application of house vacuum while the membrane is positioned on the stage could result in stripping or removing of bound analytes or antibodies on the membrane resulting in lower and uneven signals. It is appreciated that the vacuum can be optional, and that in some embodiments, the device can utilize an absorbant material (e.g. stack of absorbant sheets) that can hold all the liquid to be disposed of (i.e., the transfer buffer, water wash, pbs wash, and all the antibody and wash blotting cycles, etc.). In such embodiments, the instrument would not require a separate vacuum / pressure source.

[0140] In another aspect, the pad can be used and multiple cycles performed in order to achieve greater blotting sensitivity (3-4x) and equivalent reproducibility to the standard process. To demonstrate this, the membrane was preprepared with dot blots of humanKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO transferrin protein with 10 replicates of 6 dilutions (2-fold serial dilution from 0.2ug to 0.00625 ug) randomly distributed across the membrane. The dot blot membrane was attached to the cartridge and then the blot processing performed by probing with a rabbit anti-human transferrin monoclonal antibody and goat anti-rabbit IgG-HRP conjugate (Cell Signaling Technologies), followed by chemiluminescent detection. It was found that performing 15 cycles of withdrawing antibody at 0.2ml / min flow for 2min, followed by 20 sec blotting to the filter pad had >3x signal than the control standard process using a rocking platform with similar levels of reproducibility. The signals were lower than the control when blotting to the filter pad was omitted. A similar increase in signal with equivalent %CV were obtained when 6ml of primary antibody was first withdrawn into the cartridge and then 15 cycles performed of infusing the cartridge to push antibody out of the cartridge for 10-15 sec at 0.5 ml / min, incubating for 2min and then blotting to the filter pad for 5 sec. While a specific protocal is described here, it is appreciated that changes to the number of cycles, step durations, flow rates, direction of flow, antibody concentrations, addition of buffer amendments such as crowding agents (e.g. PEGs, dextrans), or warming reagents could be used to further optimize and improve the assay performance.

[0141] FIGS.32B-32C show experimental results of this procedure. These results show that this novel blot processing protocol that incorporates intermittent blotting to a pad both accelerates processing and increases signals compared to standard methods using a rocking platform. Though the mechanism of action is not proven, the increased signals may be related to mass transfer effects and enhanced rate of antibody binding. As the membrane is incubated with antibody solution the concentration of free antibody decreases which could slow the rate of binding. Upon blotting to the pad and then pushing a fresh aliquot of antibody solution into the pores of the membrane the binding rate can be accelerated once again. Repeating this more times achieves higher and higher signals presumably until all the binding sites have been saturated.

[0142] In another aspect, the vacuum / pressurization means can be incorporated into the blotting cartridge itself. In one such example, the cartridge can include an integrated syringe pump feature. One way to control the flow of reagents through the membrane is using positive and negative pressure from a syringe pump which can accurately control the rate and volume of fluids. In a novel embodiment the membrane cartridge could also integrate the syringe pump feature simplifying the hardware. An example cartridge is shown in the figure. The up / down movement of the gasketed plate (yellow edge) results in solution withdrawal or infusion intoKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO or out of the cartridge and past the membrane. The cartridge may have additional ports as shown on the right side to relieve pressure so that the syringe plunger can be moved or reset without causing a force on the fluids. An example of such an embodiment is shown in FIG. 26. As shown, the blotting cartridge 2600 includes a membrane cartridge body 2610, a syringe plunger drive shaft 2620 connected to a syringe plunger 2622 that is movable within the interior chamber of the cartridge to increase / decrease pressure to draw / expel solution into the chamber through the porous layer. A pressure release port 2630 can be provided in the side of the cartridge body. As in previous cartridge embodiments, the cartridge includes a porous support layer 2640 and a membrane 2650 at the bottom.

[0143] FIG. 33 shows an exemplary automated workflow 3300 for the system described herein. In some embodiments, the workflow can be completed within about two hours, which is considerably faster than conventional approaches. In this embodiment, the workflow includes steps of: 1) preparing the device 301, 2) performing electrophoresis 302, 3) performing protein transfer 303, 4) performing probe blot 304, 5) performing image blot, and 6) analyzing the image to identify targets 306. The first step of preparing the device 301 can entail the user loading samples into the gel and loading consumables inside the instrument. It is estimated this steps takes about 15 minutes or less. The second step of performing protein gel electrophoresis entails stain-free protein normalization, for example as described in any of the embodiments herein. It is estimated this step takes about 20 minutes or less. The third step of protein transfer 3203 can entail mating a blotting cartridge membrane with a top face of the gel and applying current between anode and cathode, thereby transferring the protein to the membrane. It is estimated this step takes about 10 minutes or less. The fourth step of probe blotting 3204 can entail step blotting a blotting cartridge through reagents and processing using positive and negative pressure applied via the blotting cartridge. It is estimated this steps takes about 64 minutes or less. The fifth step of image blotting 3205 can entail fluorescence or chemiluminescence. It is estimated this step takes about 10 minutes or less. The sixth and final step of analyzing the images 3206 can entail any suitable methodology and outputting the results with the system. By the above-described automated workflow, it is estimated the entire process can be completed within 1.5-2 hours, thereby drastically increasing throughput as compared to conventional methods. Western Blot results vs Traditional Shaking MethodsKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0144] FIG.34 shows the western blot results using a preferred embodiment of this invention which was completed in 2.2 hrs and compared against a Western blot performed using an abbreviated standard Western blot process using a rocking platform for processing that took about six hours to perform.50 ul of HeLa lysate (Rockland Immunochemicals) was mixed with 1.25 ul of mercaptoethanol reducing agent and heated for 5 minutes at 100 ºC to denature proteins. For the control, a 12% Protean TGX mini-gel was loaded with 5 ug of heated lysate (lanes 2-7, 9-11) or 5 ul of Precision All Molecular Weight Standard (Bio-Rad) (lanes 1, 8, 12) and run for distance of about 3cm. After SDS-PAGE the proteins in the gel were transferred to a Trans-Blot Turbo PVDF membrane per instructions. The control membrane was processed using a rocking platform throughout. Blocking was for 60min using PBST + BSA. Primary antibody (mix of Rabbit anti-human pan AKT and beta-Actin monoclonal antibodies (Cell Signaling Technologies) was incubated at 1:1000 dilution in PBST + 1% BSA for 60 min with rocking, followed by four washes with PBST for 5 minutes each. Secondary antibody (1:10,000 goat-anti-rabbit IgG-HRP secondary antibody, Cell Signaling Technologies) in PBST + 1% BSA was incubated for 60 minutes with rocking and then washed five times five minutes in 1X PBST. The membrane was then incubated for 5 min with chemiluminescence substrate and imaged using a Chemidoc MP imager (Bio-Rad).

[0145] For a preferred embodiment of the present invention, the same lysate samples were loaded into wells 2-6 and 8-11 and MW standards into wells 1, 7 and 12 of a U-cast cassette gel card and electrophoresis performed for 20 minutes. The gel card was extricated from the cassette and moved to a stainless-steel electrode containing a TBT pad soaked with transfer buffer and a membrane cartridge (PVDF previously wet and redried with 1% SDS to make hydrophilic) containing 10ml of TBT transfer buffer was placed atop the gel card and 0.7 kg of force applied. Transfer was performed for 5400 Joules which required eight minutes. After transfer, the port on the top of the cartridge was attached to a tubing connected to a 50 ml syringe and a syringe pump, and the membrane was then placed atop a TBT pad resting above a vacuum filtering unit with the vacuum off. The syringe pump was set to infuse to push the transfer buffer out of the cartridge at five ml / min for two minutes. The membrane cartridge was then consecutively washed with water followed by PBS buffer using a back-and-forth flow over an eight minute period, infusing the withdrawn solution in the cartridge to the pad on the filter unit each time. The membrane was blocked with BSA for five minutes by flowing the solution back and forth through the membrane. Rabbit anti-human monoclonal antibodies (pan AKT and beta-Actin from Cell Signaling Technologies) were diluted 1:1000 in 1x PBST + 1%KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO BSA and added to one compartment of a reagent tray (6ml). Six compartments were filled with 4ml of 1X PBST, one with 10 ml of goat-anti-rabbit IgG-HRP secondary antibody (1:2000 dilution, Cell Signaling Technologies) and one with 4ml of Clarity Max Chemiluminescent substrate (Bio-Rad Labs). The membrane cartridge was then sequentially stepped through primary antibody, 3 washes, secondary antibody, three washes, and finally chemiluminescent substrate using a preferred protocol that took a total of 76 minutes to perform including blocking. The membrane was excised from the cartridge and imaged using a Chemidoc MP imager (Bio-Rad). Gel electrophoresis through image acquisition required 106 minutes.

[0146] FIG.34 shows the control blot on the left and the invention embodiment on the right. The image on the right shows a pseudo-colored overlay of the protein MW standards and the specific antibody detected AKT and beta-actin bands. The images show that the preferred embodiment has as good or better resolution and sensitivity as the control blot that took about three times as long to perform.

[0147] In yet another aspect, lateral flow can be used for increasing throughput without dramatically increasing workflow time. Some embodiments of this invention increase the sample throughput which refers to the number of samples analyzed per unit time. In some embodiments, a gel card cassette may have twelve samples and western blotting completed in about two hours. In other embodiments, two or more gels can be run at the same time doubling the throughput. In some instances, the doubling of throughput could double the total assay time. Other ways to increase throughput without linearly increasing the time would be advantageous to the user as they could generate more results per day. In some embodiments, the number of samples per gel card cassette is increased such as with 24 samples doubling the throughput without increasing the assay time. Combining different numbers of sample wells, and different hardware changes, such as incorporating multiple syringe pumps or power supplies could also allow for higher throughput devices without dramatically increasing the total assay time. Staggering the start of different steps in the assay is another common means to increase throughput in a non-linear fashion. In a different embodiment, a lateral flow device such as those described in U.S. Patents 10,688,487 and 11,826,753, the entire contents of which are incorporated herein by reference for all purposes, could be used for the blot processing steps. In this embodiment, 1 or more gels could be processed through the blocking step and then the cartridge or cartridges positioned on a lateral flow card where the antibodies and wash solutions are sequentially delivered to the membranes of each cartridge being processed. In this way, the processing steps, which are the longest duration in the automated workflow, would beKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO performed simultaneously, greatly reducing the total workflow time while increasing assay throughput.

[0148] To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0149] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted. As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise. As used herein, the terms “and / or” or “ / ” means any one of the items, any combination of the items, or all of the items with whichterm is associated.

[0150] As used herein, the word “substantially” does not exclude “completely,” e.g., a composition that is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the disclosure. The term “substantially” may also be used herein to emphasize this meaning, particularly when the described embodiment has the same or nearly the same characteristic or functionality, unless the context or other statements clearly indicate otherwise. As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection, but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0151] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” isKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment, and may be considered to include values within + / - 25% of the recited value or +- 10% of the recited value.

[0152] As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0153] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0154] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0155] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Additional Disclosure and Claimable Subject Matter

[0156] Embodiment 1. An automated system for analyzing a biological sample, the system comprising: (i) a separation module comprising: (a) a gel disposed on a support layer, whereinKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO the gel comprises a plurality of microwells, and wherein the gel is permeable to an electric field along a perpendicular axis at least within a sample separation flow path from a sample well to an opposite end of the gel; (b) a set of anode and cathode buffer reservoirs comprising running buffer; (c) one or more electrode(s) embedded in the cathode and anode buffer reservoirs; and (ii) a blotting module comprising: (a) a blotting cartridge comprising: a chamber comprising a first opening on a first side and a second opening on a second side; a transfer membrane attached directly or indirectly to the chamber and spanning the first opening of the blotting cartridge, vacuum / pressure connection to the second opening; and an anode electrode, wherein the blotting cartridge is movable between the blotting module and an upper surface of the gel in the separation module; (b) a blotting reservoir positioned under the separation module to receive transfer buffer; (c) a reagent tray; and (iii) an imaging module comprising: an image sensor positioned below the blotting cartridge, the image sensor is optionally in contact with the transfer membrane during imaging.

[0157] Embodiment 2. The system of embodiment 1, wherein the imaging module further comprises an excitation source positioned below the blotting cartridge, wherein the excitation source is configured to excite biological samples on the transfer membrane; and optionally, a filter positioned between the image sensor and the blotting cartridge, wherein the filter corresponds to a wavelength of the excitation source.

[0158] Embodiment 3. The system of any one of embodiments 1-2, wherein the gel comprises agarose, polyacrylamide, or a blend thereof.

[0159] Embodiment 4. The system of embodiment of any one of embodiments 1-3, wherein the gel is polyacrylamide.

[0160] Embodiment 5. The system of any one of embodiments 1-4, wherein the blotting cartridge comprises a support layer backing the transfer membrane.

[0161] Embodiment 6. The system of any one of embodiments 1-5, wherein the blotting reservoir contains a cathode electrode.

[0162] Embodiment 7. The system of any one of embodiments 1-6, wherein the microwells have a width of about 0.5 mm to about 5 mm and have a depth of about 0.3 to about 10 mm.

[0163] Embodiment 8. The system of any one of embodiments 1-7, further comprising a data visualization module, which comprises a computer system for analyzing emission signals from the transfer membrane.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0164] Embodiment 9. The system of any one of embodiments 4-8, wherein the polyacrylamide gel comprises about 4% to about 20% of acrylamide.

[0165] Embodiment 10. The system of any of embodiments 1-9, where the gel comprises a label.

[0166] Embodiment 11. The system of embodiment 10, wherein the label is a haloalkane selected from the group consisting of trichloroethanol, chloroform, trichloroacetic acid, trichloroethane, bromoform, and iodoacetic acid; or a UV excitable dye.

[0167] Embodiment 12. The system of any one of embodiments 5-11, wherein the support layer of the blotting cartridge comprises a porous material.

[0168] Embodiment 13. The system of embodiment 12, wherein the porous material is selected from the group consisting of polyethylene, polypropylene, cellulose, porous plastic polymers, cellulose / polymer blend, glass fibers, porous ceramics, graphite, aluminum oxide, porous foams, and combinations thereof.

[0169] Embodiment 14. The system of any one of embodiments 1-13, wherein the gel is supported between two porous films.

[0170] Embodiment 15. The system of any one of embodiments 1-14, wherein the system separates, transfers, and detects proteins in the biological sample in less than about 2 hours.

[0171] Embodiment 16. The system of any one of embodiments 1-15, wherein the transfer membrane comprises a material selected from polyvinylidene fluoride (PVDF), nylon and nitrocellulose.

[0172] Embodiment 17. The system of any one of embodiments 1-16, wherein the anode in the blotting cartridge reservoir is electrically paired with a cathode in the blotting tray reservoir.

[0173] Embodiment 18. The system of any one of embodiments 1-17, wherein the reagent tray comprises compartments pre-filled with blotting reagents.

[0174] Embodiment 19. The system of embodiment 18, wherein the blotting reagents are independently selected from blocking buffer, wash buffer, primary antibody solution, primary antibody diluent, secondary antibody solution, secondary antibody diluent, diluent, protein- conjugated reporter molecule, oligonucleotide-conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0175] Embodiment 20. The system of embodiment 18 or 19, wherein the compartments are each pre-filled with about 1 mL to about 10 mL of the blotting reagents.

[0176] Embodiment 21. The system of any one of embodiments 1-20, wherein the blotting cartridge is connected to a vacuum or pressure source and delivers each blotting reagent to the membrane when the membrane is in contact with corresponding compartments of the reagent tray.

[0177] Embodiment 22. The system of embodiment 21, further comprising a waste module coupled to the vacuum source.

[0178] Embodiment 23. The system of any one of embodiments 1-22, wherein the excitation source comprises at least one light emitting diode (LED) emitting light at a wavelength in a range from about 325 nm to about 700 nm, an ultraviolet light source, or both.

[0179] Embodiment 24. The system of any one of embodiments 1-23, wherein the image sensor is configured for colorimetric, chemiluminescent, or fluorescent detection.

[0180] Embodiment 25. The system of any one of embodiments 1-24, further comprising an x-y-z gantry attached to the blotting cartridge for controlling movement of the blotting cartridge.

[0181] Embodiment 26. A method for detecting proteins in a biological sample using the system according to any one of embodiments 1-25, the method comprising: (a) inserting the gel into a blotting reservoir, wherein the gel is optionally pre-loaded with the biological sample, molecular weight standard, and running buffer; (b) engaging a separation anode with a separation cathode in the separation module, and applying an electric current between the separation anode and cathode to separate the proteins into a plurality of protein bands; (c) dispensing transfer buffer to the blotting tray reservoir under the gel and into the blotting cartridge; (d) lowering the blotting cartridge toward the first surface of the gel until the transfer membrane contacts the first surface of the gel, engaging a transfer anode with a transfer cathode, and applying an electric current between the transfer anode and the transfer cathode to transfer the plurality of bands from the gel to the transfer membrane; (e) removing the transfer buffer from the blotting cartridge and washing the blotting cartridge with water or a wash buffer; (f) processing the transfer membrane by sequentially aspirating one or more blotting reagents from one or more individual compartments of the reagent tray through theKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO transfer membrane of the blotting cartridge to form a plurality of labeled bands on the transfer membrane; and (g) detecting an optical signal from each of the plurality of labeled bands.

[0182] Embodiment 27. The method of embodiment 26, wherein the gel is a Stain-FreeTM gel, the plurality of labeled bands are a plurality of labeled protein bands, and the method further comprises performing Stain-FreeTM imaging of the gel, wherein after step (b), the Stain-FreeTM gel comprising a plurality of protein bands is exposed to ultraviolet light to form labeled protein bands.

[0183] Embodiment 28. The method of embodiment 26 or 27, wherein steps (b) through (g) are automated.

[0184] Embodiment 29. The method of any one of embodiments 26-28, further comprising reporting results in tabular and / or graphical formats.

[0185] Embodiment 30. The method of any one of embodiments 26-29, further comprising normalizing the optical signal.

[0186] Embodiment 31. The method of any one of embodiments 26-30, wherein the optical signal is a colorimetric, fluorescent, or chemiluminescent signal.

[0187] Embodiment 32. The method of any one of embodiments 26-31, wherein the separation in step (b) occurs in about 5 minutes to about 20 minutes.

[0188] Embodiment 33. The method of any one of embodiments 26-32, wherein step (d) comprises lowering the blotting cartridge to the upper surface of the gel at a pressure from about 0.5 kg-force to about 2 kg-force.

[0189] Embodiment 34. The method of any one of embodiments 26-33, wherein the transferring the plurality of bands in step (d) occurs in about 1 minute to about 10 minutes.

[0190] Embodiment 35. The method of any one of embodiments 26-34, wherein the removing the transfer buffer from the blotting cartridge in step (e) is carried out by vacuum aspiration or pressure.

[0191] Embodiment 36. The method of any one of embodiments 26-35, wherein the washing the blotting cartridge in step (e) is performed at a water or buffer wash station, wherein the blotting cartridge is rinsed and aspirated to remove excess transfer buffer solution from the transfer membrane.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0192] Embodiment 37. The method of any one of embodiments 26-36, wherein the blotting cartridge is rinsed after aspirating each of the blotting reagents in the reagent tray in step (f).

[0193] Embodiment 38. The method of any one of embodiments 26-37, wherein the one or more blotting reagents are selected from blocking buffer, primary antibody, primary antibody diluent, wash buffer, secondary antibody, secondary antibody diluent, diluent, protein- conjugated reporter molecule, oligonucleotide-conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution.

[0194] Embodiment 39. A kit for automated analysis of proteins in a biological sample, the kit comprising: (i) one or more components selected from:(a) a precast gel;(b) a disposable blotting cartridge;(c) a reagent tray;(d) a running buffer solution; (e) a transfer buffer solution; and(f) a cleaning pad; and (ii) packaging information and instructions for use,wherein the precast gel comprises agarose, polyacrylamide, or a blend thereof.

[0195] Embodiment 40. The kit of embodiment 39, wherein the reagent tray comprises compartments pre-filled with one or more components selected from blocking buffer, wash buffer, primary antibody solution, primary antibody diluent, secondary antibody solution, secondary antibody diluent, diluent, protein-conjugated reporter molecule, oligonucleotide- conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution.

[0196] Embodiment 41. The kit of embodiment 39 or 40, wherein the precast gel has a length of about 2 cm to about 8 cm and has a width of about 2 cm to about 10 cm.

[0197] Embodiment 42. The kit of any one of embodiments 39-41, wherein the disposable blotting cartridge comprises a transfer membrane attached directly or indirectly to and spanning a first opening of a chamber in the blotting cartridge.

[0198] Embodiment 43. The kit of embodiment 42, wherein the transfer membrane is polyvinylidene fluoride (PVDF), nylon, or nitrocellulose.

[0199] Embodiment 44. A gel card for use in an automated system for analyzing a biological sample, the gel card comprising: a support frame of a generally rectangular shape and having a top and bottom plate, the support frame defining a gel slot therebetween; a plurality of microwells along a proximal edge of the support frame, wherein each of the plurality of microwells is open to the gel slot within the support frame; a gel layer disposed within the gel slot, wherein the gel is permeable to an electric field along a perpendicular axis at least within a sample separation flow path from each sample well of the plurality to an opposite end of theKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO gel in the gel slot; and a plurality of openings along the top plate extending along each separation flow path to facilitate subsequent protein transfer after electrophoresis.

[0200] Embodiment 45. The gel card of embodiment 44, wherein the gel slot includes a plurality of gel channels such that each separation flow path corresponds to a gel channel.

[0201] Embodiment 46. The gel card of embodiment 45, wherein each channel has a plus- shaped cross-sectional shape.

[0202] Embodiment 47. The gel card of embodiment 44, wherein the gel card includes a protruding connector to facilitate removal and transfer of the gel card from within a cassette.

[0203] Embodiment 48. A gel card cassette for use with a gel card as in embodiment 44, the gel card cassette comprising:a support frame of a generally rectangular shape and having a top and bottom plate, the support frame defining a slot in between top and bottom plates for the gel card, wherein the slot is open to facilitate removal of the gel card after electrophoresis; and connectors on each side of the support frame for securing and aligning the support frame for electrophoresis.

[0204] Embodiment 49. The cassette of embodiment 48, further comprising one or more ion flow dams extending laterally across the support frame to block ion flow above and / or below the cassette.

[0205] Embodiment 50. The cassette of embodiment 49, wherein the cassette includes at least one dam below the bottom plate and at least one dam above the top plate.

[0206] Embodiment 51. The cassette of embodiment 50, wherein the cassette comprises an anode and cathode.

[0207] Embodiment 52. The cassette of embodiment 51, wherein the cassette includes integrated buffer reservoirs.

[0208] Embodiment 53. An electrophoresis and transfer system comprising: an enclosure comprising: an electrophoresis region having a receptacle for holding buffer and a gel card cassette within during electrophoresis while the cassette is held relatively horizontal; a transfer region having a level support surface for a transfer sheet within a receptacle that holds buffer and an electrode disposed along or near the support surface to facilitate transfer of target proteins from the gel layer to the transfer sheet; and a gel layer transport element that translates the gel card from within the cartridge after electrophoresis to the transfer region.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0209] Embodiment 54. The system of embodiment 53, wherein the transfer region is higher in elevation than the electrophoresis region, the enclosure including a ramp extending between the regions.

[0210] Embodiment 55. The system of embodiment 54, wherein the ramp is angled or curved.

[0211] Embodiment 56. The system of embodiment 53, wherein the transport element includes an elongate element that pushes or pulls the gel card from within the cassette and into the transfer region.

[0212] Embodiment 57. The system of embodiment 56, wherein the elongate element is a blade.

[0213] Embodiment 58. The system of embodiment 53,wherein the transport element is controlled by an automated controller that actuates the transport element to move the gel layer into the transfer region after completion of electrophoresis and initiates the transfer in the transfer region.

[0214] Embodiment 59. The system of embodiment 58, wherein the controller further controls anode and cathode electrodes in each of the electrophoresis region and the transfer region.

[0215] Embodiment 60. A membrane cartridge comprising: a top cover having a bottom opening; a first port atop the top cover configured for connecting to a pressure / vacuum source; an electrode configured as an anode to facilitate protein transfer; a porous support layer over the bottom opening; and a transfer membrane.

[0216] Embodiment 61. The membrane cartridge of embodiment 60, wherein the electrode is a wire that extends through the first port or a second port in the top cover.

[0217] Embodiment 62. The membrane cartridge of embodiment 60 or 61, wherein the electrode has a high current density, wherein high current density is about 8 mA / cm2 or 16, or 25, or 34 or 42 mA / cm2 or more.

[0218] Embodiment 63. The membrane cartridge of any of embodiments 60-62, wherein the electrode is a titanium coated plate electrode.

[0219] Embodiment 64. The membrane cartridge of any of embodiments 60-63, wherein the electrode is a platinum wire.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO

[0220] Embodiment 65. The membrane cartridge of any of embodiments 60-64, wherein the electrode is a wire mesh.

[0221] Embodiment 66. The membrane cartridge of any of embodiments 60-65, wherein the top cover includes one or more support ribs across the bottom opening to support the support layer.

[0222] Embodiment 67. The membrane cartridge of any of embodiments 60-66, wherein the top cover is one piece.

[0223] Embodiment 68. The membrane cartridge of any of embodiments 60-66, wherein the top cover is two interfacing pieces.

[0224] Embodiment 69. The membrane cartridge of any of embodiments 60-66, further comprising a syringe pump.

[0225] Embodiment 70. The membrane cartridge of embodiment 60, wherein the two interfacing pieces comprise a top piece and a bottom piece, wherein the top piece includes the port and electrode and the bottom piece includes the support layer and one or more support ribs to suppore the membrane.

[0226] Embodiment 71. The membrane cartridge of embodiment 60, wherein the top cover is 3D printed.

[0227] Embodiment 72. The membrane cartridge of embodiment 60, wherein the porous layer is a rigid plate with a plurality of holes therein.

[0228] Embodiment 73. The membrane cartridge of embodiment 60, wherein the cartridge is configured for blotting.

[0229] Embodiment 74. The membrane cartridge of embodiment 60, wherein the cartridge is configured for sample preparation.

[0230] Embodiment 75. The membrane cartridge of embodiment 60, wherein the cartridge further includes an electroelution grid and is configured for electroelution.

[0231] Embodiment 76. A kit comprising:a blotting cartridge as in embodiment 60 wherein the membrane is provided to a customer in a prewet format containing the required volume of transfer buffer in a disposable tray.

Claims

KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO CLAIMS WHAT IS CLAIMED IS:

1. An automated system for analyzing a biological sample, the system comprising: (i) a separation module comprising: (a) a gel disposed on a support layer, wherein the gel comprises a plurality of microwells, and wherein the gel is permeable to an electric field along a perpendicular axis at least within a sample separation flow path from a sample well to an opposite end of the gel; (b) a set of anode and cathode buffer reservoirs comprising running buffer; (c) one or more electrode(s) embedded in the cathode and anode buffer reservoirs; and (ii) a blotting module comprising: (a) a blotting cartridge comprising: a chamber comprising a first opening on a first side and a second opening on a second side; a transfer membrane attached directly or indirectly to the chamber and spanning the first opening of the blotting cartridge, vacuum / pressure connection to the second opening; and an anode electrode, wherein the blotting cartridge is movable between the blotting module and an upper surface of the gel in the separation module; (b) a blotting reservoir positioned under the separation module to receive transfer buffer; (c) a reagent tray; and (iii) an imaging module comprising: an image sensor positioned below the blotting cartridge, wherein the image sensor is optionally in contact with the transfer membrane during imaging.

2. The system of claim 1, wherein the imaging module further comprises an excitation source positioned below the blotting cartridge, wherein the excitation source is configured to excite biological samples on the transfer membrane; and optionally, a filter positionedKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO between the image sensor and the blotting cartridge, wherein the filter corresponds to a wavelength of the excitation source.

3. The system of any one of claims 1-2, wherein the blotting cartridge comprises a support layer backing the transfer membrane, optionally, the support layer comprises a porous material.

4. The system of any one of claims 1-3, wherein the blotting reservoir contains a cathode electrode.

5. The system of any one of claims 1-4, further comprising a data visualization module, which comprises a computer system for analyzing emission signals from the transfer membrane.

6. The system of any one of claims 1-5, wherein the system separates, transfers, and detects proteins in the biological sample in less than about 2 hours.

7. The system of any one of claims 1-6, wherein the anode in the blotting cartridge reservoir is electrically paired with a cathode in the blotting tray reservoir.

8. The system of any one of claims 1-7, wherein the reagent tray comprises compartments pre-filled with blotting reagents.

9. The system of any one of claims 1-8, wherein the blotting cartridge is connected to a vacuum or pressure source and delivers each blotting reagent to the membrane when the membrane is in contact with corresponding compartments of the reagent tray.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO 10. The system of any one of claims 1-9, further comprising an XYZ gantry or robotic arm attached to the blotting cartridge for controlling movement of the blotting cartridge.

11. A method for detecting proteins in a biological sample using the system according to any one of claims 1-10, the method comprising: (a) inserting the gel into a blotting reservoir, wherein the gel is optionally pre- loaded with the biological sample, molecular weight standard, and running buffer; (b) engaging a separation anode with a separation cathode in the separation module, and applying an electric current between the separation anode and cathode to separate the proteins into a plurality of protein bands; (c) dispensing transfer buffer to the blotting tray reservoir under the gel and into the blotting cartridge; (d) lowering the blotting cartridge toward the first surface of the gel until the transfer membrane contacts the first surface of the gel, engaging a transfer anode with a transfer cathode, and applying an electric current between the transfer anode and the transfer cathode to transfer the plurality of bands from the gel to the transfer membrane; (e) removing the transfer buffer from the blotting cartridge and washing the blotting cartridge with water or a wash buffer; (f) processing the transfer membrane by sequentially aspirating one or more blotting reagents from one or more individual compartments of the reagent tray through the transfer membrane of the blotting cartridge to form a plurality of labeled bands on the transfer membrane; and (g) detecting an optical signal from each of the plurality of labeled bands, optionally steps (b) through (g) are automated.

12. The method of claim 11, further comprising reporting results in tabular and / or graphical formats.KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO 13. The method of any one of claims 11-12, further comprising normalizing the optical signal, optionally the optical signal is a colorimetric, fluorescent, or chemiluminescent signal.

14. The method of any one of claims 11-13, wherein any of : separation in step (b) occurs in about 5 minutes to about 20 minutes; and transferring the plurality of bands in step (d) occurs in about 1 minute to about 10 minutes.

15. The method of any one of claims 11-14, wherein any of: wherein removing the transfer buffer from the blotting cartridge in step (e) is carried out by vacuum aspiration or pressure; wherein the washing the blotting cartridge in step (e) is performed at a water or buffer wash station, wherein the blotting cartridge is rinsed and aspirated to remove excess transfer buffer solution from the transfer membrane; and wherein the blotting cartridge is rinsed after aspirating each of the blotting reagents in the reagent tray in step (f).

16. A kit for automated analysis of proteins in a biological sample, the kit comprising: (i) one or more components selected from: (a) a precast gel; (b) a disposable blotting cartridge; (c) a reagent tray; (d) a running buffer solution; (e) a transfer buffer solution; and (f) a cleaning pad; andKT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO (ii) packaging information and instructions for use, wherein the precast gel comprises agarose, polyacrylamide, or a blend thereof.

17. The kit of claim 16, wherein the reagent tray comprises compartments pre-filled with one or more components selected from blocking buffer, wash buffer, primary antibody solution, primary antibody diluent, secondary antibody solution, secondary antibody diluent, diluent, protein-conjugated reporter molecule, oligonucleotide- conjugated reporter molecule, chemiluminescent substrate, and membrane stripping solution.

18. A gel card for use in an automated system for analyzing a biological sample, the gel card comprising: a support frame of a generally rectangular shape and having a top and bottom plate, the support frame defining a gel slot therebetween; a plurality of microwells along a proximal edge of the support frame, wherein each of the plurality of microwells is open to the gel slot within the support frame; a gel layer disposed within the gel slot, wherein the gel is permeable to an electric field along a perpendicular axis at least within a sample separation flow path from each sample well of the plurality to an opposite end of the gel in the gel slot; and a plurality of openings along the top plate extending along each separation flow path to facilitate subsequent protein transfer after electrophoresis.

19. The gel card of claim 18, wherein the gel slot includes a plurality of gel channels such that each separation flow path corresponds to a gel channel.

20. The gel card of claim 18 or 19, wherein each channel has a plus-shaped cross- sectional shape.

21. A gel card cassette for use with a gel card as in claim 44, the gel card cassette comprising:KT Ref: 094260-1511053-123110PC Bio-Rad Ref.: BRP01264-WO a support frame of a generally rectangular shape and having a top and bottom plate, the support frame defining a slot in between top and bottom plates for the gel card, wherein the slot is open to facilitate removal of the gel card after electrophoresis; and connectors on each side of the support frame for securing and aligning the support frame for electrophoresis.

22. The cassette of claim 21, further comprising one or more ion flow dams extending laterally across the support frame to block ion flow above and / or below the cassette.

23. An electrophoresis and transfer system comprising: an enclosure comprising: an electrophoresis region having a receptacle for holding buffer and a gel card cassette within during electrophoresis while the cassette is held relatively horizontal; a transfer region having a level support surface for a transfer sheet within a receptacle that holds buffer and an electrode disposed along or near the support surface to facilitate transfer of target proteins from the gel layer to the transfer sheet; and a gel layer transport element that translates the gel card from within the cartridge after electrophoresis to the transfer region.

24. A membrane cartridge comprising: a top cover having a bottom opening; a first port atop the top cover configured for connecting to a pressure / vacuum source; an electrode configured as an anode to facilitate protein transfer; a porous support layer over the bottom opening; and a transfer membrane.

25. A kit comprising: a blotting cartridge as in claim 60 wherein the membrane is provided to a customer in a prewet format containing the required volume of transfer buffer in a disposable tray.

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