System and method for photopolymerization of electrophoresis gels

Abstract

Acrylamide gel formulations for use in electrophoresis, and systems and methods for electrophoretic gel casting. In some embodiments, the system includes a UV-Vis curing light source and an acrylamide formulation that allows for photopolymerization of the separating gel and stacking gel in a single step. In some embodiments, the separation solution formulation includes acrylamide and bisacrylamide, Bis-Tris buffer, and lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate (LAP) as a water-soluble photoinitiator, which allows the solution to be photopolymerized using UV radiation within a specific wavelength. In some embodiments, the dilution buffer formulation includes acrylamide and bisacrylamide, Bis-Tris buffer, and lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate (LAP). The dilution buffer can be added to the separation solution in a selected amount to modify the concentration of acrylamide to a desired concentration.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 236,020, filed August 23, 2021, the disclosure of which is incorporated herein by reference in its entirety. [Background technology]

[0002] The use of gel electrophoresis, such as polyacrylamide gel electrophoresis (PAGE), is a widespread technique for the separation of biological materials. While nonbiological materials can also be separated using gels or other chromatographic supports, the scope of work with biological materials is even greater. Typical applications include the separation of nucleic acid fragments of various sizes in the context of sequencing; the detection of polymorphisms; or size verification in other contexts. Gel separation is also frequently applied to the separation of proteins (e.g., by SDS-PAGE using sodium dodecyl sulfate), glycoproteins, and protein fragments, as well as for verifying homogeneity and purity, identifying post-translational modifications, and confirming molecular weight.

[0003] In all these procedures, a mixed sample of biological entities is applied to an electrophoresis gel, and the components are separated by applying an electric field across the gel. Regardless of how the gel is developed, the resulting migration pattern of the substances contained in the sample is detected, for example, by chemiluminescence or fluorescence detection.

[0004] Western blotting is a technique that involves immobilizing proteins on a membrane before detection using monoclonal or polyclonal antibodies. Before immobilizing the proteins on the membrane, sample proteins are separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate native and denatured proteins. The proteins are then transferred, or electroblotted, onto a membrane, where they are probed and ultimately detected using antibodies specific to the target protein. The specificity of the antibody-antigen interaction allows for the identification of a single protein within a complex protein mixture.

[0005] To separate proteins with similar molecular weights, SDS-PAGE is often performed in discontinuous gels, where a stacking gel is cast on top of the resolving gel. The stacking gel is more porous than the resolving gel (due to the former's lower polyacrylamide concentration), which is partially responsible for effective protein separation. The concentration of acrylamide in the resolving gel can be varied to achieve optimal protein separation.

[0006] Systems for hand-casting electrophoresis gels are commercially available and include a variety of approaches (e.g., single, dual, and multicast). Most hand-casting systems require users to set up the system by assembling a series of variable-sized glass plates with spacers appropriate for the desired gel thickness they wish to cast (e.g., 0.75 mm, 1.0 mm, 1.5 mm). The desired gel thickness is determined by the sample volume required for the electrophoresis process. To set up these casting systems, the glass plates are properly aligned and loaded into a holder or tank. This mechanically compresses the glass plates, forcing the glass against some kind of gasketing means (e.g., silicone spacers) to prevent the acrylamide liquid from leaking from the system. The compressed glass plates form a cassette with a pocket between them that is filled with liquid polyacrylamide gel before polymerization into an electrophoresis gel.

[0007] Once the casting system is assembled, gels can be cast by adding the liquid acrylamide solution to the glass cassette. Polyacrylamide gels are typically formed from two layers: a low-percentage acrylamide stacking gel, which concentrates proteins at the gel interface, and a high-percentage acrylamide separating gel, which separates proteins and other macromolecules by molecular weight. The stacking and separating gel solutions consist of a specific ratio of acrylamide monomer and a crosslinker (such as bis-acrylamide). Polymerization of acrylamide and the crosslinker into polyacrylamide gels is often achieved using the chemical initiators ammonium persulfate (APS) and tetramethylethylenediamine (TEMED). This method allows for complete polymerization of the stacking and separating gels in 1–2 hours. Alternatively, riboflavin can also be used as a polymerization initiator, often combined with TEMED as a catalyst. Riboflavin is a photoinitiator that releases free radicals when exposed to light; complete polymerization can take up to 8 hours using this method.

[0008] Once the system is properly assembled and set up, the first step in the casting process begins with introducing the liquid acrylamide separating gel into the glass cassettes, usually by pipetting. Single and dual systems require pouring into narrow openings at the top of each individual cassette. Multicast systems allow users to flood an entire stack of cassettes simultaneously, but this generates excess acrylamide waste, which must be washed away after the entire casting process is complete. Visibility within the cassette is crucial to achieving the desired height of the separating gel; this is an advantage in single and dual cassette systems, but is unachievable beyond the first few layers in multicast systems. When creating an acrylamide separating gel formulation, ammonium persulfate (APS) and TEMED are mixed with acrylamide to catalyze the polymerization of the gel. After pouring this liquid acrylamide formulation into the cassette, users must wait 30–45 minutes for the separating gel to fully polymerize, which is one of the most time-consuming steps in creating an electrophoresis gel.

[0009] Once the separating gel has polymerized, the second step of the casting process involves introducing an acrylamide stacking gel into each cassette, again by pipetting, on top of the separating gel from the first step. Once the stacking gel is in the cassette, the user inserts a sample well comb tailored to the gel's thickness; the comb consists of multiple teeth to form wells, the number of which is based on the sample size and required well volume (common configurations are 10-, 12-, and 15-well combs). Similar to separating gel formulations, stacking gel formulations typically consist of acrylamide, APS, and TEMED, but the reagent concentrations vary. A similar waiting period of 30-45 minutes is required for the stacking gel to fully polymerize.

[0010] In a typical casting method, the separating gel solution is first added to a casting cassette, and then alcohol is placed on top to prevent polymerization inhibition by oxygen. After the separating gel is allowed to polymerize for 30-60 minutes, the alcohol overlay is poured off, the cassette is gently rinsed with deionized water, and allowed to dry. Next, the stacking gel solution is added to the top of the cassette, and a sample well comb is inserted to form the wells. The completed gel is allowed to polymerize for 60 minutes, after which it can be used or stored for later use. Summary of the Invention [Problem to be solved by the invention]

[0011] In some embodiments, it is desirable to provide an improved polymerization process and system that has a simplified user workflow that reduces wait times, reduces the number of preparation steps, and / or reduces the number of toxic chemicals used. It is also desirable to provide a repeatable polymerization process and system that utilizes a gel formulation that includes a photoinitiator that allows polymerization with an appropriately selected and oriented light source. [Means for solving the problem]

[0012] Embodiments disclosed herein relate to acrylamide gel formulations for use in electrophoresis, as well as systems and methods for electrophoresis gel casting. In some embodiments, the system includes a light source, such as a UV-Vis curing light source, and an acrylamide formulation that photopolymerizes the separating gel and stacking gel in a single step. In some embodiments, the acrylamide formulation allows the separating gel and stacking gel to be photopolymerized in multiple steps. In some embodiments, the separation solution formulation includes acrylamide and bisacrylamide, a Bis-Tris buffer, an optional density modifier such as sucrose or glycerol, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a water-soluble photoinitiator, which allows the solution to be photopolymerized using radiation within a specific wavelength. In some embodiments, the dilution buffer formulation includes a bis-tris buffer, an optional density adjuster such as sucrose or glycerol, and lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate (LAP) as a water-soluble photoinitiator (which allows the solution to be photopolymerized using radiation within a specific wavelength), with or without acrylamide and bisacrylamide. The dilution buffer can be added to the separation solution in a selected amount to modify the acrylamide concentration to the desired concentration. Preferably, the density adjuster, if used, is the same in the separation solution and the dilution buffer.

[0013] In some embodiments, the stacking solution includes acrylamide and bisacrylamide, a bis-tris buffer, and lithium phenyl-2,4,6-trimethyl-benzoylphosphinate (LAP) as a photoinitiator (which also allows the solution to be photopolymerized using radiation within certain wavelengths).

[0014] In some embodiments, the separation solution and stacking solution can be formulated such that the density of the separation solution is greater than the density of the stacking solution, allowing the stacking solution to be positioned above the separation solution (e.g., upstream of the separation solution in the direction of sample migration in use) when in liquid form, such as in a gel cassette. As a result, both the separation solution and the stacking solution can be polymerized simultaneously in a single step.

[0015] In certain embodiments, an electrophoresis discontinuous gel system is disclosed, comprising a separating gel and a stacking gel, wherein the separating gel is polymerized from a separating solution formulation having a first density and comprising acrylamide and bisacrylamide, Bis-Tris buffer, sucrose, and lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate. The stacking gel is polymerized from a stacking solution formulation having a second density and comprising acrylamide and bisacrylamide, Bis-Tris buffer, and lithium phenyl-2,4,6-trimethylbenzoyl-phosphinate, wherein the first density is greater than the second density. In some embodiments, the electrophoresis gel system is formed by simultaneously polymerizing the separating solution formulation and the stacking solution formulation.

[0016] In other embodiments, the separating and stacking solutions are combined with little or no density gradient, such as by removing the density modifier in the separating gel, and the separating and stacking solutions are polymerized sequentially rather than simultaneously.

[0017] In certain embodiments, the system includes a gel cassette containing a separating solution formulation and a stacking solution formulation before polymerization. In certain embodiments, the system includes a gel cassette containing a separating gel and a stacking gel after polymerization.

[0018] In some embodiments, one or both of the separating solution and the stacking solution are polymerized by a suitable light source. In some embodiments, the electrophoresis system includes a suitable light source. In some embodiments, the suitable light source includes UV-Vis light. In some embodiments, the UV-Vis light source has a wavelength of 365 nm to 405 nm.

[0019] In some embodiments, a gel cassette is formed by two flat glass plates spaced apart by one or more suitable spacers (usually placed along the perimeter of the plates), creating a volume or pocket between the plates. The thickness of the spacer defines the thickness of the gel. The spacer may be a separate, independent component or may be integrated into or glued to one of the plates. The gel cassette acts as a gel holder, holding the gel in place during use.

[0020] In certain embodiments, an apparatus designed for rapid filling and polymerization of electrophoresis gels is disclosed.

[0021] The embodiments disclosed herein may take form in various components and arrangements of components, and in various process operations and arrangements of process operations. The drawings are only for purposes of illustrating preferred embodiments and are not to be construed as limiting. This disclosure includes the following drawings: [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic side view of a gel cassette being illuminated with a UV light source, according to certain embodiments. [Figure 2] FIG. 1 is a perspective view of a light source and a casting frame according to certain embodiments. [Figure 3] FIG. 1 is a perspective view of a housing that houses a casting frame, according to certain embodiments. [Figure 4]FIG. 3 is a perspective view of the housing of FIG. 2 showing a casting frame and gel cassette, according to certain embodiments. [Figure 5] FIG. 10 is a perspective view of a housing according to an alternative embodiment. [Figure 6] FIG. 1 is a perspective view of a gel casting system having multiple casting assemblies according to certain embodiments. [Figure 7] FIG. 1 is a perspective view of a gel casting system with a double-sided light source for gel polymerization. DETAILED DESCRIPTION OF THE INVENTION

[0023] Although specific terminology is used in the following description for clarity, these terms are intended to refer only to the particular structure of the embodiments selected for illustration in the drawings and are not intended to define or limit the scope of the present disclosure. It should be understood that in the drawings and the following description, like number designations refer to components of like function.

[0024] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0025] As used herein, various devices and parts may be described as "including" other components. As used herein, the terms "including," "comprising," "having," "having," "can," and "containing," and variations thereof, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional components.

[0026] All ranges disclosed herein are inclusive of the recited endpoints and are independently combinable (e.g., the range "2 inches to 10 inches" includes the endpoints 2 inches and 10 inches, and all intermediate values).

[0027] As used herein, approximation language may be applied to modify any quantitative expression that may vary without resulting in a change in the associated basic function. Thus, values ​​modified by terms such as "about" and "substantially" may not be limited to the exact value specified in some cases. The modifier "about" should also be considered to disclose a range defined by the absolute values ​​of the two endpoints. For example, the expression "about 2 to about 4" also discloses a range of "2 to 4."

[0028] It should be noted that many of the terms used herein are relative terms. For example, where the terms "upper" and "lower" are used, the locations are relative to one another, i.e., the upper component is located at a higher elevation than the lower component, and should not be construed as requiring a particular orientation or location of the structure. As a further example, when the terms "internal," "external," "inside," and "outside" are used, they are relative to the center and should not be construed as requiring a particular orientation or location of the structure.

[0029] When the terms "top" and "bottom" are used, this is in absolute terms, i.e., relative to the Earth's surface. In other words, an upper location is always higher towards the Earth's surface than a lower location.

[0030] The terms "horizontal" and "vertical," when used, are used to indicate orientation relative to an absolute reference, i.e., ground level, but should not be construed as requiring structures to be perfectly parallel to one another or perfectly vertical.

[0031] Referring first to FIG. 1, a schematic diagram of an exemplary system for polymerizing acrylamide gel formulations according to certain embodiments is shown. The system includes a light source 1 configured to emit light to photopolymerize an acrylamide-based solution 2, which is held within a glass cassette assembly 3 sealed with a gasket 4 beneath the solution 2 and the glass cassette assembly 3. The glass cassette assembly 3 can be of conventional design and construction, as known to those skilled in the art. For example, glass plates with one or more spacers appropriate for the desired gel thickness to be cast (e.g., 0.75 mm, 1.0 mm, and 1.5 mm) can be used. The glass plates are appropriately aligned and loaded onto a caster device, which serves to hold the cassette assembly together and mechanically compress the glass plates, forcing the glass against the spacers or gaskets to prevent acrylamide liquid from leaking from the assembly. The separation solution, dilution buffer (if used), and stacking solution can be manually injected into the glass cassette assembly 3 by methods well known in the art, such as pipetting.

[0032] According to certain embodiments, separation solution and stacking solution formulations are provided that allow for polymerization of both solutions in a single step. This is a significant time-saving improvement over conventional formulations that require the separation solution to be polymerized first, thereby allowing the stacking solution to be properly oriented on the resulting polymerized separation gel before polymerizing the stacking solution. In some embodiments, the separation solution formulation includes acrylamide and bis-acrylamide, Bis-Tris buffer (2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol), sucrose, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as a water-soluble photoinitiator (which allows the solution to be photopolymerized using radiation within a specific wavelength range). Suitable separation solution formulations include acrylamide:bis-acrylamide weight ratios between 19:1 and 37.5:1. Suitable weight ratios include: 19:1, 19.5:1, 20:1, 20.5:1, 21:1, 21. 5:1, 22:1, 22.5:1, 23:1, 23.5:1, 24:1, 24.5:1, 25:1, 25.5:1, 26:1, 26.5:1, 27:1, 27.5:1, 28:1, 28.5:1, 29:1, 29.5:1, 30:1, 30.5:1, 31:1, 31.5:1, 32:1, 32.5:1, 33:1, 33.5 :1, 34:1, 34.5:1, 35:1, 35.5:1, 36:1, 36.5:1, 37:1, 37.5:1, and any ratio within the aforementioned ranges (e.g., 19.75:1). Preferably, the total acrylamide concentration is 8-20% w / v, the Bis-Tris concentration is 200-375 mM, and the LAP concentration is 0.015% w / v.

[0033] The concentration of acrylamide in the separation solution can be customized by combining the separation solution with a dilution buffer, where the dilution buffer has a lower acrylamide concentration than the separation solution and includes a Bis-Tris buffer, sucrose or other density adjuster, and LAP. An exemplary dilution buffer can have a total acrylamide concentration of 0-10% w / v, a sucrose concentration of 4-10% w / v, a Bis-Tris concentration of 200-375 mM, and an LAP concentration of 0.015% w / v.

[0034] In some embodiments, the stacking solution comprises acrylamide and bisacrylamide, a Bis-Tris buffer, and lithium phenyl-2,4,6-trimethyl-benzoylphosphinate (LAP) as a photoinitiator (which also allows the solution to be photopolymerized using radiation within a specific wavelength). A suitable stacking solution formulation includes an acrylamide:bis-acrylamide weight ratio of 19:1, a total acrylamide concentration of 4.5-5.5% w / v, a Bis-Tris concentration of 375 mM, and a LAP concentration of 0.0015-0.05% w / v.

[0035] In this embodiment, the separation solution and stacking solution are formulated so that the density of the separation solution is greater than the density of the stacking solution. This allows the stacking solution to be placed above the separation solution without mixing (e.g., upstream of the separation solution in the direction of sample migration during use); when in a liquid state, such as in a gel cassette, the denser separation solution supports the less dense stacking solution, forming an interface between the two. As a result, both the separation solution and the stacking solution can be polymerized simultaneously or in a single step, rather than the traditional two-step polymerization.

[0036] In certain embodiments, a density gradient between the separation solution and the stacking solution is created by including a density modifier in the separation solution formulation. An exemplary density modifier is sucrose. A suitable amount of sucrose in the separation solution ranges from 4 to 10% w / v. Another exemplary density modifier is glycerol in a similar amount.

[0037] In another embodiment, there is little or no density gradient between the separation solution and the stacking solution, such as by elimination of a density modifier. In this embodiment, the acrylamide formulation is comprised of the separation solution formulation, the diluent buffer formulation, and the stacking solution formulation, except that no density modifier is present.

[0038] In certain embodiments, the separating and stacking solutions are oriented vertically within the glass gel cassette assembly 3, such that upon polymerization the resulting stacking gel is above the resulting separating gel. This orientation ensures that proteins migrate downward through the stacking gel and into the separating gel during electrophoresis.

[0039] FIG. 2 shows an embodiment of an assembly having a light source 1, a gel cassette 2, and a casting frame 10. The light source 1 should be positioned relative to the gel cassette 2 to achieve optimal light intensity to effectively irradiate and polymerize the acrylamide gel solution held within the gel cassette assembly. For optimal interaction between the radiation and the polyacrylamide gel, the light must be completely unobstructed as it illuminates the gel window within the glass cassette assembly 2. The only exception is the clear glass between the light and the gel. The surface uniformity of the light as it interacts with the gel should be 90% or greater. In a specific embodiment, the light source should be positioned approximately 1 inch to 3 inches from the gel cassette assembly 2. The optimal light intensity range at the aforementioned distance is 6 mW / cm. 2 ~23mW / cm 2 To activate the photoinitiator, the wavelength of the light source must be between 365 nm and 405 nm. Suitable wavelengths include 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, and 405 nm.

[0040] 3 and 4 show an exemplary housing 20 that may be used to house a light source 1, such as an LED array, having the particular light parameters described above and maintaining the same position while illuminating the gel. The housing may include a controller 21 that is placed in communication with the light source and may be configured to control various operating parameters such as illumination time, light intensity, light pulses, etc.

[0041] The one or more controllers used in any of the embodiments disclosed herein can have a processing unit and a storage element. The processing unit can be a general-purpose computing device such as a microprocessor. Alternatively, it can be a specialized processing device such as a programmable logic controller (PLC). The storage element can utilize any memory technology, such as RAM, DRAM, ROM, flash ROM, EEROM, NVRAM, magnetic media, or other media suitable for retaining computer-readable data and instructions. The controller unit can be in electrical communication (e.g., wired, wireless) with one or more operating units in the system, including the UV light source. The controller can also be associated with a human-machine interface (HMI) that displays or prompts an operator for one or more parameters involved in the operation of the system and / or the execution of the methods described herein. The storage element can include instructions that, when executed by the processing unit, enable the system to perform the functions described herein. In some embodiments, multiple controllers can be used. The controller 21 can be detachable from the housing 20.

[0042] In certain embodiments, the housing 20 includes a cassette holder 22 disposed within the housing for holding a gel cassette, allowing a user to clearly view the gel during the casting operation and providing convenient access to the cassette with hands or a pipette during the casting operation.

[0043] In some embodiments, the housing 20 has a movable door 24 that is movable between a closed position ( FIG. 3 ) and an open position ( FIG. 4 ), the latter position exposing and providing access to the gel cassette. In the embodiment of FIG. 4 , the light source 1 can be coupled to the inside surface of the door 24, such that closing the door 24 properly orients the light source relative to the gel cassette, and does so in a repeatable and reproducible manner. This allows full exposure of the gel to light without interfering with its illumination. A safety mechanism can be included to prevent the light from being turned on when the door 24 is open.

[0044] In certain embodiments, a removable positioning tray or platform 23 is associated with the housing and oriented within the housing to capture any chemical spills that may occur during operation, and may be removable for ease of cleaning. The removable tray 23 may have a positioning mechanism 25 for positioning the gel cassette assembly and a positioning mechanism (e.g., magnets) (not shown) for itself within the housing 20, thereby allowing for repeatable and reproducible positioning of the casting frame 10 and gel cassette assembly 3 within the housing.

[0045] FIG. 5 shows an additional embodiment of the curing system. In this embodiment, the positioning tray 23 and light source 1 with controls are separate entities. To create gels using this embodiment, the housing 20 is first removed, followed by placing the casting frame 10 with the gel cassette assembly 3 on the tray 23 before the gel cassettes are filled with separating and stacking gels. The housing is then positioned to accommodate the casting frame 10, cassette assembly 3, and tray 23. As with the embodiment of FIG. 4, the light source is coupled to the interior surface of the housing 20, and once the housing is properly oriented relative to the gel cassette assembly 3, the light source is also properly oriented. This embodiment allows for a more compact footprint and the possibility of creating a modular platform with the tray 23, allowing a user to fill one cassette with gel while photopolymerizing another gel.

[0046] FIG. 6 illustrates yet another embodiment of a modular curing system. In this embodiment, multiple casting assemblies are oriented in a back-to-back relationship, each with its own dedicated light source. As with the previous embodiment, each light source 1 may be a single-sided panel attached to an interior wall of the housing 20″, such that when the housing is properly positioned relative to the gel cassette assembly, each light source is properly oriented relative to the gel cassette assembly for optimal light illumination. In the illustrated embodiment, the first and second light sources 1 are positioned on opposite interior walls of the housing 20″ such that light emitted from the first light source is directed toward the second light source.

[0047] Figure 7 shows yet another modular embodiment in which multiple casting assemblies (not shown) are arranged side-by-side, allowing the light source for each casting assembly, such as via a double-sided light panel containing an array of LED lights on each side, to be positioned between the two assemblies as shown.

Claims

1. 1. An electrophoresis gel formulation comprising: a. acrylamide and bisacrylamide; a buffer comprising 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; Lithium phenyl-2,4,6-trimethyl-benzoylphosphinate as a water-soluble photoinitiator a separating gel comprising: b. Acrylamide and bisacrylamide; a buffer comprising 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol; Lithium phenyl-2,4,6-trimethyl-benzoylphosphinate as photoinitiator a stacking gel comprising: Here, the density of the separating gel is greater than the density of the stacking gel.

2. 10. The electrophoresis gel formulation of claim 1, wherein the weight ratio of acrylamide:bisacrylamide in the separating gel is from 19:1 to 37.5:

1.

3. 3. The electrophoresis gel formulation of claim 2, wherein the total acrylamide concentration in the separating gel is 8-20% w / v.

4. 3. The electrophoresis gel formulation of claim 2, wherein the concentration of the buffer in the separating gel is 200-375 mM and the concentration of the photoinitiator in the separating gel is 0.015% w / v.

5. 10. The electrophoresis gel formulation of claim 1, wherein the weight ratio of acrylamide:bisacrylamide in the stacking gel is 19:

1.

6. 3. The electrophoresis gel formulation of claim 2, wherein the total acrylamide concentration in the stacking gel is 4.5 to 5.5% w / v.

7. 3. The electrophoresis gel formulation of claim 2, wherein the concentration of the buffer in the stacking gel is 375 mM and the concentration of the photoinitiator in the stacking gel is 0.025% w / v.

8. 10. The electrophoresis gel formulation of claim 1, wherein the separating gel further comprises a density modifier.

9. 9. The electrophoresis gel formulation of claim 8, wherein the density modifier is sucrose or glycerol.

10. 2. The electrophoresis gel formulation of claim 1, wherein the weight ratio of acrylamide:bisacrylamide in the separating gel is from 19:1 to 29:

1.

11. A system for polymerizing an electrophoresis gel formulation, comprising: a. a gel cassette configured to hold a gel formulation; wherein the gel formulation comprises a polymerizable separating gel solution and a polymerizable stacking gel solution; The polymerizable separating gel solution contains acrylamide and bisacrylamide, a buffer containing 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol, and lithium phenyl-2,4,6-trimethyl-benzoylphosphinate as a water-soluble photoinitiator; the polymerizable stacking gel solution comprises acrylamide and bisacrylamide, a buffer comprising 2,2-bis(hydroxymethyl)-2,2′,2″-nitrilotriethanol, and lithium phenyl-2,4,6-trimethyl-benzoylphosphinate as a photoinitiator; the density of the polymerizable separating gel solution is greater than the density of the polymerizable stacking gel solution; b. A light source having a wavelength of 365 to 405 nm.

12. The light source had an output of 6 mW / cm at the surface of the gel formulation. 2 ~23mW / cm 2 12. The system of claim 11, wherein the light source is oriented relative to the gel formulation to have a light intensity of

13. The system of claim 11 , wherein the polymerizable separating gel solution further comprises a density modifier.

14. The system of claim 13 , wherein the density adjuster is glycerol or sucrose.

15. 12. The system of claim 11, wherein the weight ratio of acrylamide:bisacrylamide in the polymerizable separating gel solution is 19:1 to 37.5:

1.

16. A method of casting an electrophoresis gel, comprising: a. formulating a separating gel solution comprising acrylamide and bisacrylamide, a buffer comprising 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol, and lithium phenyl-2,4,6-trimethyl-benzoylphosphinate as a water-soluble photoinitiator; b. Formulating a stacking gel solution comprising acrylamide and bisacrylamide, a buffer comprising 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol, and lithium phenyl-2,4,6-trimethyl-benzoylphosphinate as a photoinitiator; wherein steps a and b are performed such that the density of the separating gel solution is greater than the density of the stacking gel solution; c. introducing the separating gel solution into a gel cassette; d. introducing the stacking gel solution into the gel cassette above the separating gel solution; and e. The separating gel solution and the stacking gel solution are simultaneously exposed to UV light at a wavelength of 365 nm to 405 nm to polymerize the separating gel solution and the stacking gel solution.

17. 17. The method of claim 16, further comprising the step of varying the concentration of acrylamide in the separating gel solution by adding to the separating gel solution a dilution buffer comprising 0-10% w / v total acrylamide, 2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol, sucrose, and lithium phenyl-2,4,6-trimethyl-benzoylphosphinate.

18. The UV light source has a power of 6 mW / cm 2 ~23mW / cm 2 17. The method of claim 16, wherein the separating gel solution and the stacking gel solution are irradiated with a light intensity of

19. 17. The method of claim 16, wherein the weight ratio of acrylamide:bisacrylamide in the separating gel solution is 19:1 to 37.5:1.

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