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Method for marking protein

A protein and labeling technology, applied in the field of biochemistry, can solve the problems of poor lysine modification selectivity and low solubility of modified substrates, and achieve the effect of significant technological progress.

Pending Publication Date: 2022-02-18
SHENZHEN BAY LAB PINGSHAN TRANSLATIONAL MEDICINE CENT +2
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Problems solved by technology

[0004] Aiming at the above-mentioned problems in the prior art, the present invention provides a method for labeling proteins. The method for protein labeling needs to solve the problems of poor selectivity of lysine modification, modification of substrates, and Problems such as low solubility of substances

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  • Method for marking protein
  • Method for marking protein

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Embodiment 1

[0024] 1. Cultivate the A549 cell line in a 10cm cell culture dish until its growth density reaches 80%-90%, discard the medium, add 2mL PBS to wash three times, carefully scrape the cells from the culture dish with a cell scraper, and collect In 2mL ep tube. Use 40% power to sonicate the cells, stop 1s every 1s, and sonicate for 5min.

[0025] 2. Centrifuge at 13000rpm for 10min, collect the supernatant, take 1μL of BSA of 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL, 5mg / mL and 1μL of the supernatant and place them in a 96-well plate 150 μL of Coomassie Brilliant Blue was added, and the absorbance at 490 nm was measured using a multifunctional microplate reader. Then draw a standard curve of protein concentration and absorbance value, and convert the protein concentration in the supernatant.

[0026] 3. Dilute the protein concentration to 2mg / mL with boric acid buffer solution with pH=10, take 1mL of the lysate and add S-dimethyl-2-propargylsulfonium salt with a final concentration of 5...

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Abstract

The invention discloses a method for marking protein. The method comprises the following steps: selecting a protein needing to be marked, using a functional molecule with carboxyl or thiocarboxyl as a substrate, using unsaturated sulfonium salt as a carboxyl activator, and carrying out a solid-phase peptide bond generation reaction on the marked protein in an alkaline buffer solution; in a buffer solution, carrying out amine ester exchange reaction activated by unsaturated sulfonium salt and carrying out specific modification reaction on amino groups on the protein; and identifying the specific modification condition by means of western blot or mass spectrum. Functional molecules containing carboxyl or thiocarboxyl are used as substrates, unsaturated sulfonium salt is used as a carboxyl activating agent, and in different types of buffer solutions, different types of various proteins can be marked through amine ester exchange reaction. According to the method, safe and low-toxicity sulfonium salt activated ester is used as a labeling molecule, protein can be labeled efficiently and selectively, and the method can be used for activity-based protein analysis and labeling of antibody molecules in a drug-coupled antibody.

Description

technical field [0001] The invention belongs to the field of biochemistry, and relates to a method for labeling proteins, specifically a method for covalently labeling proteins by activating carboxylic acids with unsaturated sulfonium salts in buffer solutions in different physiological environments. Background technique [0002] Covalent labeling of native proteins by site-specificity is the most commonly used method. Compared with genetic engineering, it has the advantages of shorter experimental period and less uncertain factors in the experiment, so it has become a protein labeling method favored by scientific researchers. However, due to the relatively mild environment of living organisms, not all organic chemical reactions are suitable for protein labeling. Therefore, the following points need to be paid attention to: first, the chemical reaction of protein labeling must be resistant to biological environmental conditions (pH 6-8, ≤37 ° C, aqueous solvent); second, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/13C07K1/107C07K1/08C07K1/04G01N27/62
CPCC07K1/13C07K1/1075C07K1/086C07K1/04G01N27/62
Inventor 李子刚尹丰万川冯源王蕊孔凌微
Owner SHENZHEN BAY LAB PINGSHAN TRANSLATIONAL MEDICINE CENT
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