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Co-immunoprecipitation test method based on ELISA method

A technology of co-immunoprecipitation and test method, which is used in the quantitative detection of plate-type solid-phase co-immunoprecipitation-enzyme-linked assay, rapid screening and quantitative detection of co-precipitated proteins, and can solve the problem of complex operation process, low sensitivity, and inaccessibility. Results and other issues, to achieve the effect of improving experimental efficiency, simplifying the experimental process, and reducing the experimental cost

Inactive Publication Date: 2008-05-28
JIANGSU UNIV
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Problems solved by technology

But this method still has three obvious defects: first, the combination of two proteins may not be a direct combination, but a third party may act as a bridge in the middle; second, the method itself is risky, and the target protein must be predicted before the experiment. What, to select the antibody for final detection, so if the prediction is incorrect, the

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  • Co-immunoprecipitation test method based on ELISA method

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Embodiment

[0027] 1.1 Cell culture

[0028] Human glioma cell line U251 was purchased from the Shanghai Institute of Cell and Biochemistry, Chinese Academy of Sciences, cultured in medium (DMEM, GIBCO) containing 10% calf serum (GIBCO) at 37°C in 5% CO 2 Cells were cultured, and when the cells were confluent, 2×10 5 Inoculated into 24-well plates at a concentration of 1 / ml, 0.5ml per well. 37°C 5% CO 2 After culturing for 48 hours, the cells were replaced with serum-free medium to culture the cells for 24 hours, and the cells were replaced with DMEM containing 100 ng nerve growth factor (NGF) to continue culturing the cells for 1 hour.

[0029] 1.2 The co-localization of phosphorylated tyrosine kinase A (pTrkA) and nerve growth factor (NGF) in cells was determined by immunofluorescence double labeling technique.

[0030] In order to prove that the above two proteins may interact in cells, firstly, immunofluorescence double-labeling technique was used to determine whether phosphorylate...

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Abstract

The invention discloses a method for testing co-immunoprecipitations, which is based on an ELISA method and relates to biological techniques. The invention combines a co-immunoprecipitation enzyme method and a linked immunosorbent assay for detection method. The invention comprises using a 96-holes plastic plate which is in the linked immunosorbent assay for detection method to catch a compound of protein (protein A), interesting protein (protein B), and anti interesting protein antibody, then using horseradish peroxidase which is corresponded with the anti interesting protein antibody to mark two antis, coloring, and testing through an enzyme mark instrument. The invention simplifies experimental process, and increases sensibility of a testing system. The invention has characteristics of stability and reproducibility, and the invention lowers experimental cost and increases experimental efficiency.

Description

technical field [0001] The present invention relates to biotechnology, in particular to a co-immunoprecipitation test method based on the ELISA method, specifically to a rapid, high-throughput, quantitative detection of protein-protein interaction co-immunoprecipitation-enzyme-linked test technology, To establish a quantitative detection method for plate solid phase co-immunoprecipitation-enzyme-linked assay for rapid screening and quantitative detection of co-precipitated proteins. Background technique [0002] At present, there are many methods to study the interaction between proteins, such as yeast two-hybrid system, immunoprecipitation, pull-down and other methods are commonly used. Among them, Co-Immunoprecipitation (Co-Immunoprecipitation) is a relatively stable and simple method, and it is also a method that has been widely used in the scientific research of proteome. It is based on the specific interaction between antibodies and antigens. The classic approach to st...

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N21/31
Inventor 龚爱华张志坚孙湘兰步雪峰肖德生杨勇王穆兵谷晓楚
Owner JIANGSU UNIV