Method for detecting protein interaction by immunological coprecipitation based on protein chip and reagent kit for detecting protein interaction

A protein interaction and co-immunoprecipitation technology, applied in the field of protein interaction detection, can solve the problems of complicated operation process, cultivating a large number of cells, limited antibodies, etc. Effect

Inactive Publication Date: 2008-01-16
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

[0004] 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 has some obvious shortcomings: such as complicated and cumbersome operation, time-consuming

Method used

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  • Method for detecting protein interaction by immunological coprecipitation based on protein chip and reagent kit for detecting protein interaction
  • Method for detecting protein interaction by immunological coprecipitation based on protein chip and reagent kit for detecting protein interaction
  • Method for detecting protein interaction by immunological coprecipitation based on protein chip and reagent kit for detecting protein interaction

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preparation example Construction

[0050] Preparation of the Flag antibody chip: Paste a film on the aldehyde-based glass slide to form 3 × 6 chip frames for differentiating samples. Spray the anti-flag M2 monoclonal antibody and mouse IgG (as a control antibody) into each partition frame of the aldehyde-based glass slide, and dot 3 points containing anti-flag M2 monoclonal antibody in parallel in each reaction frame and the corresponding number of Spots containing mouse IgG. Store at 4°C for later use.

[0051] 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 used.

[0052] Construction of bait and prey expression vectors: construct the coding gene of the bait on the flag tag vector, and construct the coding gene of the prey on the Myc tag vector to form flag-...

Embodiment 1

[0055] Example 1: Co-immunoprecipitation chip analysis of the interaction between p65 and p50 subunits of nuclear transcription factor-κB (NF-κB)

[0056] The p65 and p50 subunits of NF-κB exist in the form of heterodimers under physiological conditions, therefore, this example is designed with the interaction between p65 and p50 as positive.

[0057] 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) into each partition frame of the aldehyde-based glass slide, and each reaction Three spots containing anti-flag M2 monoclonal antibody and three spots containing mouse IgG were spotted in the frame. Store at 4°C for later use.

[0058] 2. Construction and identification of bait and prey expression vectors: p65 (NM_021975) and p50 (NM_003998) w...

Embodiment 2

[0105] Example 2: Verification of the interaction of six pairs of potentially interacting proteins by the co-immunoprecipitation chip:

[0106] In this example, six pairs of proteins derived from yeast two-hybrid were verified for interaction by using a co-immunoprecipitation chip.

[0107] In the early stage, TRB3 protein (triblles 3) was used as bait to screen the human liver cDNA library by yeast two-hybrid, and six prey proteins were obtained: FN1, Myo18A, ATF5, ATF4, MCM3AP and HLA-B.

[0108] The bait was constructed on the pFLAG-CMV-2 eukaryotic expression vector (please refer to the method in Example 1), and the six preys were respectively constructed on the pCMV-Myc eukaryotic expression vector (please refer to the method in Example 1).

[0109] Referring to the method in Example 1; Six kinds of pCMV-Myc-prey proteins were co-transfected with pFLAG-TRB3 respectively in HEK-293 cells, and simultaneously six kinds of pCMV-Myc-preys were co-transfected with pFlag-CMV-2 a...

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Abstract

Protein-protein interaction (PPI) is important for all biological processes; therefore, the research of protein interaction is always a research hot in the fields of cell biology and molecular biology. The co-immunoprecipitation (CoIP) is a classic method which is used to research protein interaction based on the specific action between antibody and antigen and is the commonest and most effective method to find and testify PPI; the traditional CoIP steps based on a resin are complicated and labor consuming and are hard to realize the flux detection. The key point in the invention is that CoIP is implemented on glass aldehyde sheet of the fixed anti-label antibody by utilizing the epitope label of recombinant protein; protein-protein interaction (PPI) is detected by the antibody labeled by fluorescence. The method greatly simplifies the operation steps of the traditional CoIP, greatly improves the flux of sample detection and is a novel technique platform to research PPI simply, conveniently and swiftly and with high flux.

Description

Technical field: [0001] The invention relates to the field of biology, in particular to a detection method for protein interaction. Background technique: [0002] Immunoprecipitation (immunoprecipitation, IP) is a method developed by using the specific combination of antigen and antibody (for example, the specific combination of bacterial protein "Protein A" and immunoglobulin FC fragment). For example, if antibody A to protein X is used to immunoprecipitate X, protein Y bound to X in vivo may also be precipitated. Based on the physiological interaction with protein X, the immunoprecipitation of protein Y is called co-immunoprecipitation (Co-IP). [0003] Co-immunoprecipitation is the most effective and commonly used method to discover or verify the physiological interaction between two proteins. Its specific operation is to harvest and lyse the cells under the condition of maintaining the protein-protein interaction (PPI), immunoprecipitate the target protein from the cel...

Claims

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Application Information

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IPC IPC(8): G01N33/539G01N21/64G01N33/577
Inventor 刘琼明林从何为许丹科
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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