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High-flux visible chip detecting method of protein-protein interaction and detecting kit for protein-protein interaction

A protein interaction and chip detection technology, applied in the biological field, can solve the problems of limiting the number of testing samples, cultivating a large number of cells, prolonging the experimental process, etc., and achieve the effect of reducing experimental cost, simplifying the experimental process, and good repeatability

Inactive Publication Date: 2009-09-23
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] It is undeniable that traditional Co-IP, as the most classic and effective method for detecting and identifying PPIs, has played a huge role in functional genomics research. However, this method itself also has some obvious shortcomings: such as the operation process is complicated and It is time-consuming, laborious, and often requires a large number of cultured cells; in addition, the separation of immunoprecipitates by SDS-PAGE and the detection of interactions by Western blot further prolong the experimental process. These defects greatly limit the number of detection samples, which is why it The main reason why it cannot be used as a high-throughput detection method

Method used

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  • High-flux visible chip detecting method of protein-protein interaction and detecting kit for protein-protein interaction
  • High-flux visible chip detecting method of protein-protein interaction and detecting kit for protein-protein interaction
  • High-flux visible chip detecting method of protein-protein interaction and detecting kit for protein-protein interaction

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preparation example Construction

[0064] Preparation of the Flag antibody chip: Paste a film on the aldehyde-based glass slide to form 3 × 6 chip frames for differentiating samples. In each chip frame, spray anti-flag M2 monoclonal antibody and mouse IgG (as a control antibody) onto the aldehyde-based glass partition frame respectively, and dot three anti-flag M2 monoclonal antibodies in parallel in each reaction frame. Antibody and corresponding number of spots containing mouse IgG (forming a 2×3 array). Store at 4°C for later use.

[0065] In the embodiment of the present invention, the M2 monoclonal antibody was used. Since the M2 monoclonal antibody is a mouse IgG, mouse IgG was used as the antibody control. The present invention is not limited to the use of M2 monoclonal antibody, and other types of monoclonal antibodies can also be selected.

[0066] Construction of bait protein and prey protein expression vectors: the coding gene of bait protein was constructed on the flag tag vector, and the coding g...

Embodiment 1

[0069] Example 1: Co-immunoprecipitation chip analysis of the interaction between p65 and p50 subunits of nuclear transcription factor-κB (NF-κB)

[0070] The p65 and p50 subunits of NF-κB exist in the form of heterodimers under physiological conditions. Therefore, this example is designed based on the interaction between p65 and p50 as a positive model.

[0071] 1. Preparation of the Flag antibody chip: Paste a film on the aldehyde-based glass slide to form 3×6 chip frames in partitions. Use a microarray spotter (microarrayer, CAPITALBIO Boao, Jingxin SmartArrayer-48) to spray anti-flag M2 monoclonal antibody and mouse IgG (as a control antibody) onto the aldehyde-based glass slide frame in each chip frame In each reaction frame, 3 dots containing anti-flag M2 monoclonal antibody (upper row) and 3 dots containing mouse IgG (lower row) formed a 2×3 dot matrix. Prepare at least two Flag antibody chips and store them at 4°C for future use.

[0072] Two, the construction and id...

Embodiment 2

[0118] Example 2. Using the high-throughput visualization chip detection technology of the present invention to verify the interaction of 19 pairs of proteins with potential interactions, and compare the detection effect with the detection method of fluorescently labeled antibodies

[0119] In order to check the reliability of the high-throughput visualized silver-enhanced chip detection method of the present invention, 19 pairs of proteins with potential interactions (see Table 1) were randomly selected to verify with the high-throughput visualized chip detection technology of the present invention, and simultaneously adopted Fluorescence immunoassay method was tested, and each group of experiments carried a known positive interaction pair Flag-p50 / c-Myc-p65 and a known negative interaction pair Flag-Jun / c-Myc-lacZ as positive controls and negative and compare the results of the two sets of experiments.

[0120] The bait was respectively constructed on the pFLAG-CMV-2 eukaryo...

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Abstract

The invention discloses a high-flux visible chip detecting method of protein-protein interaction and a detecting kit for protein-protein interaction. The method comprises the steps as follows: a Flag antibody chip is prepared; cell lysis solution with Flag-bait fused protein and Myc-quarry fused protein is added onto the surface of the chip for co-immunoprecipitation; and existence of interaction between the bait protein and the quarry protein is detected by the combination of colloidal gold and the silver enhancement colour technology. The kit comprises an aldehyde galssslide with a plurality of sub-areas, a FLAG, a c-Myc label carrier, a monoclonal antibody corresponding to the c-Myc label vector, chain-avidin colloidal gold, silver enhancement solution A and B, and a specification based on the method. The high-flux visible chip detecting technology of protein-protein interaction established in the invention is an efficient and effective tool for proteomics research.

Description

technical field [0001] The invention relates to the field of biology, in particular to a method for detecting protein-protein interaction and a kit for detecting protein-protein interaction. Background technique [0002] Protein-protein interaction (PPI) is crucial to all biological processes. Therefore, the study of protein interaction has always been a hot research topic in the field of cell biology and molecular biology. [0003] According to the type of markers, immunoassay methods can be divided into enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, chemical or bioluminescence immunoassay, etc. Metal immunoassay is a class of immunoassay methods based on metal markers. Early metal immunoassays mostly used metal chelates to label antigens or antibodies. Since a protein molecule can only be labeled with a few metal chelates, the sensitivity of this type of method is greatly limited, usually only reaching nmol level. In order to overcome this shortcoming, ...

Claims

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Application Information

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IPC IPC(8): G01N33/566G01N33/577G01N33/543C12N15/79
Inventor 刘琼明陈卿许丹科何为
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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