Preparation method of sample for identifying specific protein acetylation modification
A protein and acetylation technology, which is applied in the field of identification of specific protein acetylation modification samples, can solve the problems that cannot be modified by protein acetylation, and achieve the effect of enhancing the success rate
Active Publication Date: 2021-07-23
右江民族医学院
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However, the above methods are all based on the study of lysine acetylation modification at the proteomic level, and cannot be used to detect the acetylation modification of a specific protein
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[0032] In a preferred embodiment of the present invention, the identification of the acetylation modification site of TLR9 is taken as an example for illustration.
[0033] A preparation method for identifying specific protein acetylation modified samples, comprising the following steps:
[0034] 1) It is determined that TLR9 has acetylation modification through endogenous experiments, and the acetyltransferase CBP of TLR9 is determined through exogenous experiments;
[0035] Among them, the endogenous experiment is specifically: using the same concentration of TLR9 receptor activator ODN (0.5μM) to stimulate mouse macrophage Raw264.7 at different time points (0min, 15min, 30min), and on the basis of ODN stimulation for 30min The above cells were stimulated by adding a sirtuin inhibitor (Trichostatin A, TSA), and then the cells were collected for immunoprecipitation (immunoprecipitation, IP) and western blot (western blot, WB) analysis. The experimental results were as follows...
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The invention belongs to the technical field of biotechnology, and particularly discloses a preparation method of a sample for identifying specific protein acetylation modification. The preparation method comprises the following steps: 1) selecting a protein A with acetylation modification and acetylated transferase B; 2) preparing a protein sample of the co-transfected plasmid A and plasmid B and a protein sample of the single transfected plasmid A; (3) selecting a protein sample of a co-transfected plasmid A and a protein sample of a co-transfected plasmid B and a protein sample of a single transfected plasmid A to finish immunoblotting analysis together; and 4) running the rest of protein samples co-transfected with the plasmid A and the plasmid B into SDS-PAGE gel, incubating in coomassie brilliant blue dye liquor overnight, decolorizing, cutting off a target strip, and putting into a centrifugal tube, thereby obtaining the acetylation modified sample for identifying the specific protein. According to the protein sample prepared by adopting the method, the success rate of identifying the specific protein acetylation modification site by the mass spectrum can be remarkably enhanced.
Description
【Technical field】 [0001] The invention relates to the field of biotechnology, in particular to a preparation method for identifying specific protein acetylation modification samples. 【Background technique】 [0002] Protein post-translational modification (PTM) refers to the chemical modification of protein after translation, which is involved in almost all normal life activities of cells and plays a very important regulatory role. As a way of protein function regulation, PTM is crucial to protein structure and function. Studies have shown that 50%-90% of proteins in the human body undergo post-translational modifications. More than 400 post-translational modifications have been identified so far, and common modifications include methylation, phosphorylation, ubiquitination, acetylation, glycosylation, sumoylation, nitrosylation, and oxidation. Therefore, PTM has become an extremely important field of protein research in the world. Among them, protein acetylation is a ubiq...
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IPC IPC(8): G01N1/28G01N1/30G01N27/62G01N33/68
CPCG01N1/28G01N1/30G01N27/62G01N33/6803
Inventor 王居平彭慧农安娜黄月艳高洁
Owner 右江民族医学院