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A kind of micropreparative gel electrophoresis device and using method thereof

A gel electrophoresis and preparative technology, applied in the field of bioanalysis and biomedical research, can solve the problems of high cost of one-time gel, long elution time, short separation gel, etc., and achieve easy reuse and humanized operation , short time effect

Active Publication Date: 2017-04-19
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These shortcomings limit the application of large-volume preparative gel electrophoresis systems in general biological sample separation analysis and proteomics
Recently, the commercialized GELFREE 8100 preparative electrophoresis instrument launched by Expedeon in the United Kingdom also uses gel electrophoresis technology to separate and collect fractions to achieve the purpose of electrophoresis preparation, but the instrument itself has too short separation gel, low resolution and long elution time And disadvantages such as high cost of disposable gel

Method used

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  • A kind of micropreparative gel electrophoresis device and using method thereof
  • A kind of micropreparative gel electrophoresis device and using method thereof
  • A kind of micropreparative gel electrophoresis device and using method thereof

Examples

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Effect test

Embodiment 1

[0046] The present invention is used to separate, elute and collect bovine serum albumin, β-casein, horse myoglobin and α-lactalbumin mixture (hereinafter referred to as sample protein), and to study its properties. Among them, the preparation method of the sample protein is as follows: weigh about 500 μg each of bovine serum albumin, β-casein, horse myoglobin and α-lactalbumin, mix them, add purified water, 5×SDS-PAGE loading buffer (250 mM Tris-HCl (pH=6.8), 10% (w / v) SDS, 0.5% (w / v) bromophenol blue, 50% (v / v) glycerol) and β-mercaptoethanol, prepared as 10 μg / μL standard protein mixture solution, which contains 1×SDS-PAGE loading buffer and 1% β-mercaptoethanol, denatured by heating in a boiling water bath for 10 minutes, and placed at -20°C spare.

[0047] Such as Figure 9 As shown, the sample protein was separated using a preparative gel electrophoresis separation device. Prepare 12% polyacrylamide gel separating gel and 4% polyacrylamide gel stacking gel respectivel...

Embodiment 2

[0051] Such as Figure 12As shown, the preparative gel electrophoresis separation device 1 of the present invention is used to separate proteins in Bacillus subtilis LM 4-2 fungal cells. The Bacillus subtilis LM 4-2 fungal cells were disrupted by an ultrasonic instrument, the total fungal protein was extracted, the protein concentration was concentrated to 10-20 μg / μL, the protein was thermally denatured, and then loaded on a preparative gel electrophoresis separation device. With embodiment 1. It can be seen that the present invention can separate actual biological samples according to different protein molecular weights.

[0052] Such as Figure 13 As shown, the separated gel is eluted and collected by using the preparative gel electrophoresis elution collection device 2 of the present invention. It can be seen that the protein bands of each component of the whole fungal protein on SDS-PAGE are arranged according to the molecular weight, indicating that the present invent...

Embodiment 3

[0054] The invention is used to separate and elute the sample protein, and calculate the recovery rate. First weigh 55 μg of bovine serum albumin (BSA), add purified water, 5×SDS-PAGE loading buffer (250mM Tris-HCl (pH=6.8), 10% (w / v) SDS, 0.5% (w / v ) bromophenol blue, 50% (v / v) glycerol) and β-mercaptoethanol to prepare a 5 μg / μL standard protein solution containing 1×SDS-PAGE loading buffer and 1% β-mercaptoethanol. After the standard protein solution was heated and denatured in a boiling water bath for 10 minutes, it was separated and collected by preparative gel electrophoresis separation device 1 and preparative gel electrophoresis elution collection device 2 respectively. After collection, each fraction was lyophilized and redissolved to 200 μL.

[0055] Then, prepare 200 μL of BSA aqueous solutions with standard concentrations of 0.1 μg / μL and 0.5 μg / μL, and load them on liquid chromatography (chromatographic column: C18 reversed-phase chromatographic column; mobile ph...

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Abstract

The invention relates to a micro gel electrophoresis device and a use method thereof. The micro gel electrophoresis device is characterized by comprising a preparation-type gel electrophoresis separation device and a preparation-type gel electrophoresis elution collection device, wherein the preparation-type gel electrophoresis separation device comprises two clamp plates, two gel preparation glass plates, a base and a refrigerating device; the two gel preparation glass plates are arranged in grooves formed in the inner side surfaces of the two clamp plates respectively; the depths of the two grooves are less than the thicknesses of the two gel preparation glass plates; the two clamp plates are placed with the inner side surfaces opposite, locked via screws and then arranged in the base in an inserting manner; the refrigerating device comprises two refrigerating pieces; the two refrigerating pieces are arranged on the outer sides of the two clamp plates respectively, and parallel to vertical planes of the two clamp plates; the preparation-type gel electrophoresis elution collection device comprises two clamp plates, an electrode cover and a collection groove; the two clamp plates are placed with the inner side surfaces opposite, and locked via screws; the electrode cover is arranged above the two clamp plates; and the collection groove is formed below the two clamp plates. The micro gel electrophoresis device can be widely used for proteomics analysis and medicine protein separation and purification.

Description

technical field [0001] The invention relates to the field of bioanalysis and biomedicine research, in particular to a micro-preparation gel electrophoresis device and its application method. Background technique [0002] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technology, as a mode of colloid electrophoresis technology, is widely used in the research of biochemistry, forensic science, molecular biology, proteomics and protein drugs, etc. and analysis fields. The principle of SDS-PAGE technology is to use the difference in molecular weight of proteins to separate them in the electrophoresis gel. The most commonly used protein separation analysis and preparation techniques are gel electrophoresis and electrophoretic elution. Among them, preparative gel electrophoresis can not only reduce the complexity of samples, enrich low-abundance proteins, remove high-abundance proteins, and separate and enrich post-translationally modified proteins before s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
Inventor 钱小红张养军郝斐然
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA