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Cell membrane receptor protein identification method and verification method

A technology of receptor protein and membrane receptor, which is applied in the field of identification of cell membrane receptor protein, can solve the problems of low efficiency, heavy workload, and unsatisfactory identification effect of membrane receptor protein

Active Publication Date: 2021-05-18
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the embodiment of the present invention is to provide a cell membrane receptor protein identification method, aiming to solve the problems of protein conformation change, low efficiency, high false positive rate, heavy workload, etc. Severe background interference leads to unsatisfactory technical problems in the identification of membrane receptor proteins

Method used

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  • Cell membrane receptor protein identification method and verification method
  • Cell membrane receptor protein identification method and verification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Modification of rLZ-8 on the gel;

[0045] Co-cultivate the modified gel with lymphocytes for 1 hour to obtain a gel with lymphocytes bound to the surface;

[0046] Quickly pipette the cell-gel mixture 10 times with a blue pipette tip, then centrifuge at 200rpm for 2 minutes, discard the supernatant containing cells and collect the beads, and resuspend the gel with normal saline;

[0047] Repeat the previous step 2 times to obtain a gel in which cells are eluted and removed;

[0048] The gel beads were treated with enzymatic hydrolysis, and then the peptides were detected by UPLC-ESI-QTOF technology, and the protein was identified by searching the protein database, and it was determined that the receptor of rLZ-8 on the surface of lymphocytes was PSAP.

Embodiment 2

[0050] Modification of rLZ-8 on the gel;

[0051] The modified gel was co-cultured with lymphocytes for 2 hours to obtain a gel with lymphocytes bound to the surface;

[0052] Quickly pipette the cell-gel mixture 10 times with a blue pipette tip, then centrifuge at 400rpm for 1 minute, discard the supernatant containing cells and collect the beads, and resuspend the gel with normal saline;

[0053] Repeat the previous step 3 times to obtain a gel from which the cells are eluted;

[0054] The gel beads were treated with enzymatic hydrolysis, and then the peptide was detected by nano-UPLC-orbitrap technology, and the protein was identified by searching the protein database, and it was determined that the receptor of rLZ-8 on the surface of lymphocytes was PSAP.

Embodiment 3

[0056] Modification of rLZ-8 on the gel;

[0057] The modified gel was co-cultured with lymphocytes for 1.5 hours to obtain a gel with lymphocytes bound to the surface;

[0058] Quickly pipette the cell-gel mixture 10 times with a blue pipette tip, then centrifuge at 300rpm for 1.5 minutes, discard the supernatant containing cells and collect the beads, and resuspend the gel with physiological saline;

[0059] Repeat the previous step 3 times to obtain a gel from which the cells are eluted;

[0060] The gel beads were treated with enzymatic hydrolysis, and then the peptides were detected by MALDI-QTOF technology, and the protein was identified by searching the protein database, and it was determined that the receptor of rLZ-8 on the surface of lymphocytes was PSAP.

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Abstract

The invention is applicable to the technical field of biology, and provides a screening method and a verification method of receptor protein on a cell membrane. The screening method comprises the following steps of modifying at least one ligand on a separation carrier, co-culturing the modified separation carrier and cells of a receptor to be screened to obtain a separation carrier of which the surface is combined with cells, eluting and separating the cells from the separation carrier based on the action of mechanical force, discarding the cells and collecting the separation carrier, and performing enzymolysis treatment on the collected separation carrier to obtain a peptide fragment, and detecting the peptide fragment to identify the membrane receptor protein. According to the method for screening the receptor protein on the cell membrane, the receptor protein is directly drawn from the surface of the cell by utilizing high binding force between the ligand and the receptor and using a mechanical elution method, so that the receptor on the cell membrane is screened, and the method is simple, efficient, good in reproducibility, and the problems that information is easy to lose, background protein interference is serious and the like in the process of extracting membrane protein by breaking cells in the existing receptor screening technology are effectively solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cell membrane receptor protein identification method and verification method. Background technique [0002] Analysis of membrane proteins has long been a challenge in protein research due to their hydrophobicity, complex post-translational modifications (PTMs), and low abundance. Commonly used methods for identifying membrane protein receptors include: extracting total cell protein or membrane protein, performing affinity chromatography pull-down or co-immunoprecipitation to catch receptor proteins for identification; in addition, yeast two-hybrid and membrane protein chips can also be used , Membrane protein RNA-sequence analysis, 2-D electrophoresis and other methods, use protein interaction to identify candidate receptors. [0003] However, the extraction of total protein or membrane protein often requires the use of detergents, resulting in changes in protein confo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 孙熙麟黄伟李鸿睿梁重阳李柏志戴璐孙非
Owner JILIN UNIV
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