Method for enriching N-phosphorylated protein
A phosphorylated protein enrichment technology, applied in the field of protein enrichment, can solve the problems of thermal instability and low natural abundance, and achieve the effects of short cross-linking time, simple and easy-to-obtain raw materials, and easy operation
Active Publication Date: 2020-06-23
XIAMEN UNIV
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Problems solved by technology
Due to the characteristics of N-phosphorylation modification, such as acid, heat instability, and low natural abundance, there has been a lack of effective enrichment and research tools, but its role in organisms is very important, so it is necessary to develop a new simple Enrichment method for further in-depth study of N-phosphorylated proteins
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[0028] The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.
[0029] The present invention mainly provides a method for immobilizing substrate protein with magnetic nanoparticles connected with a bifunctional linker, and then enriching N-phosphorylated protein under the action of a cross-linking agent. Protein arginine phosphorylated kinase McsB And its substrate protein CtsR as a model, the specific steps include: amination modification of magnetic nanoparticles; immobilization of functional protein McsB; and the use of paraformaldehyde cross-linking to connect similar prolines to the substrate The protein CtsR was enriched, and finally verified by denaturing polyacrylamide gel electrophoresis.
[0030] The embodiment of the present invention specifically includes the following steps:
[0031] 1) Bifunctional linker (H 2 N-CH 2- CH 2 -S-S-CH 2 -CH 2 -COOH) synthesis: 1.13g 3-mercaptopropionic acid and 0.8...
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Abstract
The invention discloses a method for enriching N-phosphorylated protein, and relates to protein enrichment. Magnetic nanoparticles connected with a bifunctional connecting agent are used for immobilizing functional protein, substrate protein is crosslinked under the action of a crosslinking agent, and the purpose of enriching phosphorylated protein is achieved. The preparation method comprises thefollowing steps: 1) synthesizing a bifunctional connecting agent H2N-CH2-CH2-S-CH2-S-CH2-CH2-COOH; 2) activating carboxyl-COOH on the magnetic nanoparticles; 3) connecting the magnetic nanoparticleswith a connecting agent; 4) activation of carboxyl-COOH on the coupling agent; 5) connection of phosphorylated kinase McsB protein; 6) crosslinking paraformaldehyde with phosphorylated kinase McsB andsubstrate protein CtsR thereof; and 7), separating the enriched protein with magnetic nano-particles; the method has the advantages that the raw materials are simple and easy to obtain, the crosslinking time is short, the operation is simple and convenient, a high-speed centrifugal machine is not needed in the separation process, and the whole process can be completed at room temperature.
Description
technical field [0001] The present invention relates to protein enrichment, in particular to a method for enriching N-phosphorylated protein. Background technique [0002] The combination of magnetic nanoparticles and magnetic separation technology is widely used in the enrichment and separation of biomolecules. After proper surface modification, magnetic nanoparticles can bind target proteins with high selectivity, and have high advantages in solving the problem of fast separation of proteins and high specificity of selective separation. Due to the characteristics of N-phosphorylation modification, such as acid, thermal instability, and low natural abundance, there has been a lack of effective enrichment and research tools, but its role in organisms is very important, so it is necessary to develop a new simple Enrichment methods for further in-depth research on N-phosphorylated proteins. [0003] At present, the most commonly used methods for the enrichment of traditional...
Claims
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Login to View More IPC IPC(8): G01N1/28G01N27/447
CPCG01N1/28G01N27/44778
Inventor 赵玉芬王跃桥蔡华欢
Owner XIAMEN UNIV