34 results about "IEF - Isoelectric focusing" patented technology

Described herein is a method and system for on-line coupling of capillary isoelectric focusing (cIEF) to high-resolution mass spectrometry in which a sheath flow buffer comprising polar organic solvent and organic acid is used as both an immobilization solution for (cIEF) and an ionization solution for electrospray ionization (ESI).

A method for analyzing a fluid sample is provided which includes providing a plurality of fluid samples to be analyzed and inputting each one of the plurality of fluid samples into corresponding ones of a plurality of capillaries defined within a substrate. The method further includes applying a positive charge to each one of the plurality of fluid samples at a first end of each one of the plurality of capillaries and a negative charge to each one of the plurality of fluid samples at a second end of each one of the plurality of capillaries. The method also includes transmitting an infrared light through each one of the plurality of fluid samples at a substantially same time and detecting the infrared light transmitted through each one of the plurality of fluid samples. The method further includes generating an absorption map capable of being displayed as at least one data point based on the detection of the infrared light transmitted through each one of the plurality of fluid samples.

The invention discloses a method for acquiring a two-dimensional strawberry electrophoresis differential protein map. The method comprises the following steps: (1) extracting proteins; (2) performing first-dimensional IPG isoelectric focusing electrophoresis; (3) balancing an adhesive tape; (4) transferring the adhesive tape and performing SDS-polyacrylamide gel electrophoresis; and (5) performing gel scanning and image analysis. An inventor considers the characteristic that strawberry leaves are rich in polysaccharide polyphenol substances in the invention process, the strawberry leaf protein is subjected to repeated precipitation extraction by virtue of acetone, the lysate formula is improved, the centrifugal rate, the cleaning frequency of crude protein precipitations and concentration of urea in lysate are improved, and a set of differential two-dimensional electrophoresis proteomics method suitable for strawberry leaves is constructed, so that the high-quality strawberry protein differential map is obtained. The method can be used for obtaining the two-dimensional strawberry leaf protein electrophoresis gel map with clear protein points, a large number of proteins, uniform protein distribution and a clear background.

A specified proton concentration in a volume (80) is produced by passing a controlled electrophoresis current through an adjacent electrophoresis volume (28) between a working electrode (26) and a counter electrode (24). An array of such volumes with specified proton concentration is used to provide the pH gradient for isoelectric focusing.

An elastomeric cell is used as the disposable part of an apparatus for isoelectric focusing in free solution (without gels) in the 0.5 to 5 ml volume range. An inlet port is used for priming the cell and end-connectors for coupling with electrodes. A grid of parallel rods compresses the cell against a cold plate, thereby causing swelling of the skin of the cell between pairs of rods and forming contiguous fluid bubble-compartments for IEF separation. Before collection of separated fractions, the gap between the rods and the plate is further reduced so as to create distinct fluid compartments which now contain discrete products of separation. The separated fractions are collected by syringe-like devices by puncturing the elastomer skin. The plate, rods and deformable cell are capable of rotation or gentle rocking motion around the cell's main axis to avoid gravitational convection during the focusing process.

The invention discloses a polypeptide mixture isoelectric focusing separation method for proteomics analysis. The method comprises the following steps: performing restriction enzyme digestion on a total protein mixture of a biological sample completely to obtain relatively short peptide fragment mixtures; re-dissolving the peptide fragment mixtures by using a peptide fragment isoelectric focusing buffer solution; performing isoelectric focusing electrophoresis in a loading manner by adopting a loading cup, so as to realize the effective separation of a complex peptide fragment mixture; performing desalination on a taken polypeptide mixture pre-separated from each groove and performing mass spectrometry analysis so as to obtain more polypeptide signals, higher peptide fragment covering rate and higher protein identification number compared with those obtained when a conventional method is adopted. The method is precise, efficient and low in cost, can be used in multidimensional liquid chromatography-mass spectrum identification technology systems of total proteins of the various biological samples, can replace a conventional ion exchange chromatography pre-separation method so as to realize the efficient pre-separation of the peptide fragment mixtures, and avoids the lost of peptide fragments, and therefore, proteome expression profile information with the high covering rate can be established.

An elastomeric cell is used as the disposable part of an apparatus for isoelectric focusing in free solution (without gels) in the 0.5 to 5 ml volume range. An inlet port is used for priming the cell and end-connectors for coupling with electrodes. A grid of parallel rods compresses the cell against a cold plate, thereby causing swelling of the skin of the cell between pairs of rods and forming contiguous fluid bubble-compartments for IEF separation. Before collection of separated fractions, the gap between the rods and the plate is further reduced so as to create distinct fluid compartments which now contain discrete products of separation. The separated fractions are collected by syringe-like means by puncturing the elastomer skin. The plate, rods and deformable cell are capable of rotation or gentle rocking motion around the cell's main axis to avoid gravitational convection during the focusing process.

A multiplexed, absorbance-based microfabricated chip electrophoresis system, microfabricated chip, and method of use are included for the detection and identification of protein species. The microfabricated chip electrophoresis system capable of analyzing multiple samples simultaneously. The system uses a microfabricated chip having a sample chamber for first dimension separation of a sample, a first planar array of channels for second dimension separation of the sample approximately perpendicular the sample chamber, and a barrier separating the sample chamber from the first planar array of channels during the first dimension separation. In use, the sample is placed into a sample chamber, a barrier is established around the sample chamber, the sample is isoelectric focused within the sample chamber, the barrier is then removed, and the sample separated in the first array of channels by molecular weight.

The invention relates to a method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF), belonging to the field of bioanalysis. The method comprises the steps of: firstly carrying out isoelectric focusing on carrier ampholyte solution by adopting constant voltage in a capillary to lead ampholyte with continuous pH gradient to be formed on the inner wall of the capillary; secondly injecting sample solution to the capillary and carrying out isoelectric focusing by adopting the continuous voltage; then adopting immobilized atmospheric pressure to push the sample from focusing section inside the capillary to pass a detecting window at equal speed so as to determine focus point position of the sample; and finally figuring out pH value of the focus point according to linear relation between the pH gradient and the length of the capillary. The method can be applied to the simultaneous online detection of various microorganisms and has universality for microorganism isoelectric point detection.

The invention discloses a preparation method of a micro-fluidic chip interface for two-dimensional protein electrophoretic separation, and relates to a micro-fluidic chip. The method comprises the following steps of: preparing polyacrylamide gel in a vertical channel of the micro-fluidic chip and filling a buffering aqueous solution; introducing an organic phase through a horizontal channel of the micro-fluidic chip and filling the horizontal channel; introducing an organic phase into which tetraisopropyl titanate is dissolved and filling the horizontal channel; draining an organic solution of tetraisopropyl titanate out of the horizontal channel, and cleaning the horizontal channel; adding an isoelectric focusing buffer solution into which proteins and amphoteric electrolytes are dissolved into the horizontal channel, applying electric fields to the two ends of the horizontal channel for performing isoelectric focusing, and realizing first-dimension separation of proteins in the horizontal channel according to respective different isoelectric points; and after finishing isoelectric focusing, applying electric fields to the two ends of the horizontal channel for performing second-dimension gel electrophoresis, making micropores on a TiO2 film pass through an electric field, and making the proteins enter a second-dimension channel by passing through the micropores on the TiO2 film under the action of the electric field for performing two-dimension separation.

The invention relates to a method for concentrating and desalting a urine sample prior to two-dimensional polyacrylamide gel electrophoresis, belonging to the technical field of biological detection. Certain procedures are improved on the basis of the conventional method, and the method for concentrating and desalting the urine sample comprises the following steps of: firstly, performing ultracentrifugation and organic solvent extraction; then, performing dialysis bag desalting and organic solvent extraction. In this way, the salinity is obviously reduced in comparison with the salinity obtained by using the original treatment method. Moreover, the focusing quantity in subsequent isoelectric focusing can reach 60000 vhr, which is beneficial to the successful completion of isoelectric focusing, and the picture forming quality of SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is improved.

The invention relates to a microfluidic chip, particularly to a microfluidic chip for isoelectric focusing separation, which is provided with two substrates that are bonded in reversible or non-reversible way, wherein a micro-channel and an access hole are machined in one substrate; the access hole serves a liquid storage tank; the micro-channel is provided with an extracting area and an expanding area; a transitional connecting area is arranged between the extracting area and the expanding area; and the connecting area is provided with an inclination angle. Under the premise that the pH gradient is a set value, the cyclical variation of the width of the micro-channel is utilized to enable the electric field distribution in the micro-channel to regularly vary, so as to form the non-linear pH gradient, as a result, the protein samples with similar isoelectric points can be separated in higher resolution. The microfluidic chip has the characteristics that the microfluidic chip has small size, saves reagent, is quick to separate, is convenient for integration and automation, can machine thousands of repeated micro-structures once for all, has low cost, is simple and convenient to make and operate, has high efficiency, and has excellent effect and practical significance in the isoelectric focusing separation.

Various gel electrophoretic assemblies and techniques are disclosed for providing unique isoelectric focusing (IEF) strategies. Several particular systems, assemblies and methods are provided that significantly reduce processing time, enable the use of reduced operating voltages, and produce analytical results with improved resolution.

The invention discloses a non-ampholyte free-flow isoelectric focusing electrophoretic separation method. The method comprises the steps that 1, a driving pump is used for pumping an operation flowingphase into a separation cavity of a free-flow electrophoretic device and keeping stable operation of the flow phase; 2, an electrode liquid circulation pump is used for pumping positive electrode liquid in an electrode liquid storage tank into a positive electrode chamber of the free-flow electrophoretic device and keeping circulation of the electrode liquid and used for pumping negative electrode liquid in the electrode liquid storage tank to a negative electrode chamber of the free-flow electrophoretic device and keeping circulation of the electrode liquid; 3, voltage is applied to two electrodes and adjusted to the proper value, stable operation of the device is kept, so that the device is located at the rear portion of the separation cavity, and the stable pH gradient is automaticallyformed between the electrodes. The method has the advantages that the method is simple and easy to implement, the price of the required reagent is low, and sample detection after separation cannot beinfluenced.

A device for fluid phase electrophoresis, particularly solution phase isoelectric focusing, components thereof, and methods for use are presented.

The invention discloses an isoelectric focusing electrophoretic method of hemopoietin. A BIO-RAD 111 type electrophoresis apparatus is used to carry out isoelectric focusing electrophoresis. Each 5 mLof glue comprises 1 mL of acrylamide monomer solution (25 wt%), 3.33 mL of urea (10.5 M), 0.412 mL of cane sugar (60wt%), 0.183 mL of amphoteric electrolyte solution (40 wt%, pH 2-4), 0.075 mL of amphoteric electrolyte solution (40 wt%, pH 3-10), and a catalyst solution, wherein the catalyst solution is mixed in advance and comprises 25 [mu]L of FMN-Na (0.1 wt%), 7.5 [mu]L of AP (10 wt%), and 1.5[mL] of TEMED. Before sample injection, pre-electrophoresis is performed for 10 minutes under a voltage of 200 V. Electrophoresis is performed through following phases: first phase, 50 V, 30 minutes;second phase, 100 V, 60 minutes; third phase, 200 V, 30 minutes; fourth phase, 450 V, 60 minutes; and fifth phase, 590 V, until the current is zero. The method can separate the hemopoietin charge isomers with different isoelectric points, and has the advantages that the requirement on the upper limit of voltage is not high and no cooling apparatus is needed.

The invention discloses a method for extracting soluble protein from discarded tobacco leaves. The method includes the steps: crushing the tobacco leaves into powder, and mixing the powder and extracts to prepare uniform slurry; adjusting filtered slurry to reach alkalinity to keep the slurry for a certain time at constant temperature; performing standing to obtain flocculating protein precipitation; washing and purifying protein; spray-drying the protein to obtain a soluble protein finished product. The method for extracting the soluble protein from the discarded tobacco leaves is simple, convenient and rapid, traditional steps preventing a lot of extraction such as high-speed or ultra-speed refrigerated centrifugation, gel filtration, isoelectric focusing and ultra-filtration are abandoned, protein extraction rate is larger than 65%, the protein after crude extraction is washed and purified, so that special smell of the soluble protein and parts of impurities in the discarded tobaccoleaves can be effectively removed, the purity of the soluble protein is improved, the method is suitable for large-scale industrial production, and broad prospects are provided for products such as animal feed developed by the soluble protein in the discarded tobacco leaves.

The invention provides a method for improving experiment accuracy by an isoelectric focusing method of two-dimensional protein electrophoresis, in particular a method for calculating electric energy needed by salt and protein focusing by measuring electric conductivity of a protein object to be measured. The method is characterized in that: electric energy needed by the protein focusing and salt mobility is calculated through a group of formulas in environments of different electric conductivities of the salt, the protein weight, the pH gradient gel length and the range of the pH value respectively, proper electric energy of the experiment of the individual protein object to be measured can be provided in the isoelectric focusing method so as to present an optimal protein focusing result,and the influence of colloidally protein focusing due to insufficient or over-high electric energy is avoided.

An apparatus and method are disclosed for the precise selection and extraction of a selected analyte in a focused zone produced by isoelectric focusing performed in micro-channels. A cross-channel microfluidic device comprises a sample mixture introduction and separation channel and an extraction channel, which are in fluid communication with each other at a point of intersection. Means are provided for selectively moving the pattern of separated zones following cIEF to the intersection point, and means are provided for applying an extraction pressure to direct a single zone containing a selected analyte into and then out of the extraction channel for collection.

The invention discloses an isoelectric focusing electrophoresis apparatus, comprising a positive electrode-end system, a negative electrode-end system and a focusing tank. By means of a manner of operating a plurality of sub-block mode focusing tanks and a single whole-block focusing tank, the number of adhesive tapes operated in the electrophoresis apparatus reaches 13 to 36, thus greatly improving the operation efficiency; a great number of substances can be separated and purified in batches as few as possible, so that result errors brought by variation of multiple influence factors between multiple batches are reduced, and the stability is improved; meanwhile, the efficiency of separation and purification is improved, the operation period is shortened, the cost is reduced, and the input of manpower and material resources is saved.

The present invention discloses a method for controlling electroosmotic flow in capillary isoelectric focusing electrophoresis and its equipment. It is characterized by that it adopts freeze method to freeze local fluid in the capillary to make focusing of fluid, then adopts the method of heating capillary to defreeze the solid mobile phase in the capillary and restore the transmission of fluid in the capilalry, so that the flowing-out of the sample can be batch implemented, and the focusing effect of the capillary isoelectric focusing electrophoresis can be greatly raised, and the flowing form in the column is wedge-shaped, and its separation effect is good. On the proper position of capillary separation column a heating device and a freezing device are set.

The invention provides a method for improving experiment accuracy by an isoelectric focusing method of two-dimensional protein electrophoresis, in particular a method for calculating electric energy needed by salt and protein focusing by measuring electric conductivity of a protein object to be measured. The method is characterized in that: electric energy needed by the protein focusing and salt mobility is calculated through a group of formulas in environments of different electric conductivities of the salt, the protein weight, the pH gradient gel length and the range of the pH value respectively, proper electric energy of the experiment of the individual protein object to be measured can be provided in the isoelectric focusing method so as to present an optimal protein focusing result,and the influence of colloidally protein focusing due to insufficient or over-high electric energy is avoided.

The invention provides an electrophoresis tank device for isoelectric focusing electrophoresis. By adding an electrode rotation axis to an original device and arranging an electrode end internal through hole of the electrode rotation axis at an electrode end, the horizontal swing of the electrode end during the operation of the device is effectively avoided; by arranging a plurality of flexible assemblies in an electrode end contact system, the up and down vibration during the operation of the device is effectively buffered and the effective contact between the electrode end and the surface of an adhesive tape in a focusing groove is ensured; through the buckle clamping connection of the electrode end contact system and the anti-buckle clamping connection between the electrode end contact system and a working table of an electrophoresis tank, a device service life problem caused by contact friction between the electrode end and a power supply is reduced and an adhesive tape distortion phenomenon caused by the vibration of the focusing groove during installation is also reduced. The innovative device ensures the stability of electrophoresis current to a certain extent and enhances the effect of material focus purification.

An improved method of separating a macromolecule by isoelectric focusing comprising subjecting the macromolecule to electrophoresis in an isoelectric-focusing medium including a substantially thiol-free reducing agent, preferably a trivalent phosphorous compound and more preferably tributyl phosphine, the improvement being the solubility and focusing of the macromolecule is enhanced compared to isoelectric focusing of the same macromolecule in a similar isoelectric-focusing medium containing a thiol-reducing agent.

Provided is a split electrophoresis apparatus power supply. The electrophoresis apparatus power supply and an electrophoresis apparatus are independently designed in a split mode. Modular design is adopted inside the power supply. The split electrophoresis apparatus power supply comprises a buck module, a boost module and a display operation module which are mutually connected with a motherboard. The motherboard comprises a control module and an adjusting module which are connected with each other. The split electrophoresis apparatus power supply can be compatible with traditional tubular and horizontal isoelectric focusing grooves, can be matched with the latest IPG-2-DE technology, and overcomes the inadaptability of existing products.

The invention discloses a dimensional electrophoresis method suitable for proteomic analysis of a broad bean root system. The method sequentially includes the following steps with the broad bean rootsystem as a material: isoelectric focusing; strip balancing; second direction polyacrylamide gel electrophoresis; gel staining. The broad bean root system is used as the material to establish and optimize the dimensional electrophoresis method suitable for the proteomic analysis of the broad bean root system, fill the blank of the dimensional electrophoresis technology of the broad bean root system, and provide technical support for deepening the proteomics of the underground part of the broad bean. The method is simple in step, easy to operate, good in repeatability and easy to lean and graspby technicians. All reagents used are dimensional electrophoresis conventional reagents, no expensive or rare reagent is used, and the experimental operation cost is low.

The invention discloses a preparation method of a blended ion exchange membrane and an application method for protein separation. The preparation method comprises the following steps: taking N-methylpyrrolidone as a solvent, and taking sulfonated polyaryletherketone and polysulfone as film materials for preparing a casting solution, taking an aqueous solution of isopropyl alcohol as a coagulationbath, and using a scraper to prepare the blended membrane on a nonwoven fabric. The blended ion exchange membrane is applied to an isoelectric focusing membrane electrophoresis system, a device is divided into multiple membrane chambers by the ion exchange membrane, tris(hydroxymethyl)aminomethane is taken as a buffer solution, the pH value of each membrane chambers is controlled at a certain gradient by acid-base automatic titration, and different proteins are separated into different membrane chambers by using difference of the isoelectric point of different proteins and biomolecules under electric field conditions. The blended ion exchange membrane of the present invention can achieve product purity of up to 100%.

The invention discloses a reciprocating type free flow isoelectric focusing electrophoresis device and a reciprocating type free flow isoelectric focusing electrophoresis method. The reciprocating type free flow isoelectric focusing electrophoresis device is mainly composed of a separation chamber, a liquid storage pipe, a gas liquid buffering chamber, collection pipes, recycling pipes which are located below the collection pipes and correspond to the collection pipes one by one, and a medium driving pump, wherein a separation chamber liquid outlet of the separation chamber is connected with the collection pipes and the recycling pipes respectively by switches; the liquid storage pipe is connected with a gas liquid buffering chamber liquid outlet; the gas liquid buffering chamber liquid outlet is connected with the separation chamber liquid outlet; and the medium driving pump drives media to flow in or out of the gas liquid buffering chamber from the gas liquid buffering chamber liquid inlet. According to the device and the method, one single-path driving pump is used for replacing a multi-channel driving pump; the rapid reciprocating of CA (Carrier Ampholytes) buffering liquid and a sample is used for replacing unidirectional circulation in the prior art by the separation chamber, so that the same effect of an existing RFFIEF technology is achieved; and the technology, equipment and method of the whole ReFFIEF technology are greatly simplified and the cost is reduced.