A method for identifying protein phosphorylation sites

Abstract

The invention discloses a method for identifying protein phosphorylation sites, and relates to the field of molecular biology technology. Specifically, the method for identifying protein phosphorylation sites includes separating the protein expressed by expression vector 1 through immunoprecipitation experiments , using phosphorylated antibodies to perform immunoblotting experiments to detect the modification of the phosphorylation site to be tested, the expression vector 1 contains an amino acid sequence fragment with the site to be tested, which is under the condition of no phosphorylated antibody against a single protein site , can also effectively and accurately detect the phosphorylation modification of a single site of the protein, save the cost of preparing phosphorylated antibodies for specific sites, and speed up the research process of protein phosphorylation sites.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for identifying protein phosphorylation sites. Background technique [0002] The phosphorylation and dephosphorylation process of protein regulates important life activities such as cell signal transduction, cell metabolism, cell differentiation and cell growth, and is the molecular switch of cell physiological activities. Different protein kinases can recognize and modify different sites of different proteins, which increases the complexity of the study of phosphorylated proteins, making phosphorylated proteins a hotspot in the study of protein post-translational modification. [0003] use 32 P selectable tagging of phosphorylated proteins is a classic technique in which purified kinases and 32 P for in vitro labeling, or using 32 P-ATP or 32 PO 4 3- (orthophosphate) for in vivo labeling, then the proteins were separated by SDS-PAGE, 2D-GE or thin-layer ...

AI Technical Summary

Problems solved by technology

The standard approach for phosphorylation site analysis is to use 32 P marks the purified phosphorylated protein, separates the obtained peptides by one-dimensional thin-layer electrophoresis and two-dimensional thin-layer chromatography (called two-dimensional peptide spectrum), and detects phosphorylated peptides by autoradiography. Edman degradation sequencing; or use fluorescently labeled phosphopeptides. Then identify amino acids released from phosphorylated sites by specific retention times of phosphorylated residues, release of fluorescently labeled or radioactively labeled amino acids, and this method performs large-scale When applied at scale, the workload is obviously too large

Images