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Device and method for transferring and analyzing proteins in gel on line

A protein and gel technology, applied in the field of separation and detection of proteins, can solve the problems of sample loss, difficulty in meeting the automation needs of proteomics, poor repeatability, etc., and achieve the effect of improving efficiency

Inactive Publication Date: 2013-06-19
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 2-DE (two-dimensional gel electrophoresis) and MS (mass spectrometry) are currently the most popular and reliable protein analysis platforms, but the sample processing process of proteins from two-dimensional gel to mass spectrometry is very cumbersome, and it is difficult to analyze the The isolated protein is subjected to subsequent amino acid composition and sequence analysis, mass spectrometry and other structural identification work or online qualitative and quantitative analysis
Currently reported methods that can transfer proteins in gels include direct elution, electroelution, membrane transfer, etc., which have the disadvantages of long time-consuming, cumbersome operation, poor repeatability, easy sample loss and analyte dilution, and are difficult to meet the requirements of proteomics. The need for automation in research

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  • Device and method for transferring and analyzing proteins in gel on line
  • Device and method for transferring and analyzing proteins in gel on line
  • Device and method for transferring and analyzing proteins in gel on line

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Example 1: Verification of the feasibility of transferring proteins in gels by three-electrode capillary electrophoresis

[0058] Transfer experiments were carried out from the sample simulation gel containing 0.5 mg / mL BSA and the corresponding blank simulation gel using the method of online transfer and analysis of proteins in the gel of the present invention. Among them, the role of the blank simulated gel is to compare with the sample simulated gel. By comparing the transfer electrophoresis patterns of the two, the background interference in the sample simulated gel transfer pattern is eliminated, and the peak position of the protein is determined. The results are shown in Figure 3. It can be clearly seen from Figure 3 that the peak with a peak eluting time of less than 5 minutes comes from the interference of the gel itself, and it is confirmed that the peak at 9 minutes is the BSA peak. The experimental results verified the feasibility of the method of three-elect...

Embodiment 2

[0060] Example 2: Comparison of the effect of two-electrode transfer method and three-electrode transfer method on transferring protein from gel

[0061] In 2002, Lee proposed a capillary two-electrode method for transferring proteins in a gel, that is, the outlet end of the capillary is connected to the positive electrode, and the lower end of the gel is connected to the negative electrode. In order to verify the advantages of the three-electrode transfer method proposed by the present invention, a comparative study was carried out between the two, and the transfer results of the two methods were compared using the same model sample PAGE gel, transfer voltage, transfer time and capillary and buffer conditions , the voltage across the gel was 4.5V during the three-electrode transfer. Due to the problem of reproducibility, time correction was performed on the peak area, and the ratio of the peak area to the peak time was used as the basis for judging the amount of protein trans...

Embodiment 3

[0062] Example 3: Optimization of protein transfer conditions in gels

[0063] The transfer of protein from the gel to the capillary includes two processes, one is that the protein migrates from the inside of the gel to the gel surface, and the other is that the protein enters the capillary from the gel surface. In the first process, the factors that affect the speed of protein migration are the size of the applied voltage on the gel and the size of the gel pores. One provides the driving force for protein transfer, and the other determines the resistance of proteins when they pass through the gel network. The second process is actually an electrokinetic sampling process with the help of an external electric field. Sample molecules enter the capillary under the joint action of EOF and electrophoresis. From the formula for calculating the injection volume in the electrokinetic injection method (where μ a is the electrophoretic mobility of the substance, μ EOF is the elect...

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Abstract

The invention discloses a device for transferring and analyzing proteins in gel on line. The device comprises a three-electrode power supply, a capillary, a first buffering liquid tank, a second buffering liquid tank, a detector and a metal needle tube. During transferring, the metal needle tube and the zero electrode of the three-electrode power supply are connected, are sleeved on the sample introduction end of the capillary and are placed on the upper surface of the gel; the outlet end of the capillary and the positive electrode of the three-electrode power supply are placed in the first buffering liquid tank; and the negative electrode of the three-electrode power supply is connected to the gel. During analyzing, the zero electrode of the three-electrode power supply and the sample introduction end of the capillary are placed in the second buffering liquid tank; and the outlet end of the capillary and the positive electrode of the three-electrode power supply are still placed in the first buffering liquid tank. Compared with other protein transferring methods, the device can be used for transferring and detecting simply, fast, efficiently and one line, avoid the influence of dyeing on the transferring of a protein strip in SDS-PAGE, has important theory and practical values on gel protein transferring and has very wide practical application prospect.

Description

technical field [0001] The invention relates to a technology for separating and detecting proteins, in particular to a device and method for online transfer and analysis of proteins in gel. Background technique [0002] Proteomics technology is an important method for discovering drug targets. By comparing the differences in the protein expression profiles of cells, tissues or organisms under the conditions of healthy / disease, drug-treated / untreated conditions, and identifying abnormally expressed proteins, it is possible to identify disease-related proteins and Possible targets for drug therapy. The combined application of two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) is the most commonly used method for the separation and identification of proteins in proteomics. 2-DE can separate thousands of proteins at the same time, and obtain protein expression profiles by staining. MS can accurately measure the mass of polypeptide fragments hydrolyzed by pr...

Claims

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Application Information

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IPC IPC(8): G01N27/26G01N1/28
Inventor 包建民李优鑫张勃高赫男
Owner TIANJIN UNIV
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