Monoclonal antibody capable of specifical recognization and activation of Galphaz protein and preparation method thereof

A monoclonal antibody, z protein technology, applied in the field of biological science and drug research and development, can solve the problem of inability to distinguish the activation state of Gαz protein

Inactive Publication Date: 2012-07-18
WUHAN NEWEAST BIOSCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] All existing antibodies against Gαz protein can only monitor the total Gαz protein level, but cannot distinguish the activation state of Gαz protein

Method used

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  • Monoclonal antibody capable of specifical recognization and activation of Galphaz protein and preparation method thereof
  • Monoclonal antibody capable of specifical recognization and activation of Galphaz protein and preparation method thereof
  • Monoclonal antibody capable of specifical recognization and activation of Galphaz protein and preparation method thereof

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Experimental program
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Effect test

Embodiment 1

[0025] The immunoprecipitation-immunoblotting combined method was used to prove that the monoclonal antibody of the present invention can recognize and activate the Gαz protein.

[0026] Firstly, test samples need to be prepared, which are divided into two types, adherent cell samples and suspension cell samples.

[0027] 1. Preparation of Adherent Cell Samples

[0028] (1) When the cells grow to 90% confluent density, activating or inhibiting factors are added for cell stimulation as required.

[0029] (2) Aspirate the culture medium and wash twice with pre-cooled PBS.

[0030] (3) Absorb the last PBS solution thoroughly, and add pre-cooled 1X cell lysate (0.5 ml cell lysate per 100 mm cell culture plate).

[0031] (4) Place the cell culture plate on ice for 10 minutes.

[0032] (5) Use a cell scraper to scrape the cells from the cell culture plate thoroughly.

[0033] (6) Transfer the cell lysate to an appropriate size tube and place on ice.

[0034] (7) If nuclear lysi...

Embodiment 2

[0074] ELISA method was used to quantitatively detect the content of activated Gαz protein in the test samples.

[0075] (1) Dilute and activate the monoclonal antibody of the present invention with 0.1M NaCO3 pH9.6 coating solution, coat the ELISA plate with 200 ng per well, and place it at room temperature for 2 hours or overnight at 4.

[0076] (2) Pour out the solution in the well, pat dry, add 200ulPBST to each well for washing, 3 times, 5 minutes each time.

[0077] (3) Dilute the activated Gαz protein 500 ng / ml 5-fold with diluent to 500, 100, 20, 4, 0.8, 0.16 ng / ml as a standard.

[0078] (4) At the same time prepare the test sample, cell or tissue treatment fluid supernatant.

[0079] (5) Add 50ul of the above-treated samples and standards and rabbit anti-Gαz protein antibody to each well, and let stand at room temperature for 2 hours.

[0080] (6) Pour out the solution in the well, pat dry, add 200ulPBST to each well for washing, 3 times, 5 minutes each time.

[0...

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PUM

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Abstract

The invention discloses a monoclonal antibody capable of specifically recognizing activation of Galphaz protein and a preparation method thereof, belonging to the field of bioscience and medicine development. The monoclonal antibody is generated by hybridoma which is collected in China Center for Type Culture Collection (CCTCC), and the collection number is CCTCC C2010109. The monoclonal antibody prepared by the method can specifically recognize the activation state of Galphaz protein.

Description

technical field [0001] The invention belongs to the fields of biological science and drug research and development. Background technique [0002] G protein is the main signal transduction protein in cells. The activation of Gα subunit in G protein is a sign of cell signal transduction, and it plays a key signal transduction role in cell migration and differentiation. In view of its function, this type of protein has become a major research target of cell biology and new drug discovery, that is, drug screening. According to statistics, about 70% of drugs work through the G protein pathway, through the activation of G protein and related signal channels. Detection is one of the main approaches to drug screening. [0003] G proteins have two conformations: an activated state when bound to GTP and an inactive state when bound to GDP. Normally, the vast majority of G proteins are inactive forms that bind GDP. The G protein bound to GDP can interact with a variety of receptors,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N5/16
Inventor 不公告发明人
Owner WUHAN NEWEAST BIOSCI INC
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