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Improved co-immunoprecipitation technical method

A co-immunoprecipitation and protein technology, applied in the field of optimized co-immunoprecipitation technology, can solve the problem of containing target protein and antibody

Inactive Publication Date: 2012-09-26
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, this classic technology has shortcomings in the actual operation process, that is, SDS-PAGE identifies the target protein and antibody in the isolated and purified protein.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Comparison between the improved co-immunoprecipitation method and the traditional IP method in identifying the interaction between AIF and actin

[0033] The steps are as follows:

[0034] Extraction of total intracellular protein:

[0035] 1.1 Use a petri dish with a diameter of 10 cm to culture the cells of the esophageal cancer cell line KYSE140 (a human esophageal cancer cell line from Japan, donated by Professor Shimada Y, Kyoto University), in the PRM1640 medium containing 10% fetal bovine serum, Cultivate for 3 days, and when the confluence of the cells reaches 90%, wash the cells with 4°C pre-cooled 1×PBS, 5ml / time×2 times. After washing, add pre-cooled 0.6ml cell lysate to the cell culture dish, the ingredients include 50mM Tris, pH7.4, 150mM sodium chloride, 1% triton-100 (membrane permeabilizer), 0.1 %SDS, and quickly scrape the cells with a cell scraper and collect them in a 15ml centrifuge tube. Sonication on ice (sonication for 10 seconds, wi...

Embodiment 2

[0054] Example 2: Determining Optimal Antibody and Protein G Optimal Crosslinking Time

[0055] All materials and methods are the same as in Example 1, wherein only the cross-linking time has been changed, the incubation time of step 2.3 in Example 1 is changed to: 50min, 60min and 90min, the results in figure 2 Lanes 3-5 are shown.

[0056] In step 3.4, the samples on SDS electrophoresis include the protein solution of KYSE140 whole cell lysis, the supernatant obtained from the classic IP experiment, the cross-linked 50min, 60min and 90min supernatant obtained from the improved IP experiment, and the eluted supernatant of the IP experiment After SDS electrophoresis and transmembrane transfer, conventional immunoblotting was performed using anti-AIF antibody, anti-Actin antibody and anti-heat shock protein 60 (HSP60) antibody.

[0057] The result of embodiment 2:

[0058] The total protein of the esophageal cancer cell line KYSE140 cells was subjected to the classic co-immu...

Embodiment 3

[0062] Example 3: Using the improved co-immunoprecipitation technique to identify the interaction between RIP3 protein and ENO1 The steps are as follows:

[0063] 1. Extraction of total intracellular protein:

[0064] 1.1 Use two petri dishes with a diameter of 10 cm to culture the cells of the esophageal cancer cell line KYSE140, a human esophageal cancer cell line from Japan, in the PRM1640 medium containing 10% fetal bovine serum, culture for 1 day, and wait for the fusion of the cells up to 80% degree. One plate of cells was treated with 10uM anticancer drug cisplatin for 24 hours (B), and the other plate was not treated as a control (A). Wash the cells with 4°C pre-cooled 1×PBS, 5ml / time×2 times. After washing, add pre-cooled 0.6ml cell lysate to the cell culture dish, the ingredients include 50mM Tris, pH7.4, 150mM sodium chloride, 1% triton-100 (membrane permeabilizer), 0.1 %SDS, and quickly scrape the cells with a cell scraper and collect them into a 1.5ml centrifug...

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Abstract

The invention belongs to an immunodetection field and specifically relates to an optimized co-immunoprecipitation technology. Based on an original co-immunoprecipitation technology, the co-immunoprecipitation technology is combined with a protein crosslink technology. That is to say, a protein crosslinking agent is used to crosslink an antigen antibody complex; elution is carried out by the use of a specific buffer; and the immunoblotting detection remarkably reduces the pollution of heavy chain and light chain of the antibody in the experiment. According to the invention, specificity of the co-immunoprecipitation method is raised, the method is characterized by stability and repeatability, experiment efficiency is improved, and the method provides beneficial assistance for in-depth research in molecular biology, molecular oncology, cell biology, biochemistry and the like.

Description

technical field [0001] The invention belongs to the field of immunoassay, and in particular relates to an optimized co-immunoprecipitation technique. Based on the original co-immunoprecipitation technology, the present invention combines the co-immunoprecipitation technology with the protein cross-linking technology. That is, the protein cross-linking agent is used to cross-link the antigen-antibody complex, and after elution with a specific buffer, the western blot detection significantly reduces the contamination of the antibody heavy chain and light chain in the experiment, and increases the specificity of the method. The present invention improves the specificity of the co-immunoprecipitation method, has the characteristics of stability and repeatability, improves the experimental efficiency, and provides for in-depth research on molecular biology, molecular oncology, cell biology, biochemistry and other disciplines. helpful help. Background technique [0002] With the...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 赵晓航许杨刘芳乔媛媛马首智
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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