Test method of palmitoylated modified protein based on specific antibody

Abstract

The invention belongs to the field of biochemical analysis and relates to a high-throughput test method of palmitoylated modified protein in complex biological samples based on prepared protein palmitoylated modified generic antibody serum and utilizing the same. The method includes: adopting a palmitoylated modified peptide segment as an antigen to prepare high-specificity palmitoylated modified generic antibody serum; utilizing the specific antibody serum to test protein palmitoylated modifying state. Compared with existing throughput analysis methods, the test method has the advantage of high specificity, is low in false positive rate, can directly indicate palmitoylated modified protein, can be used for samples of various sources, can identify palmitoylated modified protein in the biological samples in a high-throughput manner and is simple, convenient and easy to operate. The method provides an effective means for high-throughput testing of protein palmitoylated modifying in the biological samples.

Description

technical field [0001] The invention belongs to the field of biochemical analysis, and relates to a method for high-throughput detection of palmitoylated modified proteins, in particular to a method for detecting palmitoylated modified proteins based on specific antibodies. Modified pan-antibody and its use in the detection of biological samples. Background technique [0002] The prior art discloses that palmitoylation is one of the important forms of protein post-translational lipid modification, and plays an important role in cell signal transduction, metabolism, apoptosis, the occurrence and development of diseases, etc., among which, usually saturated The 16-carbon palmitate is covalently modified to the sulfhydryl group of the protein Cys through a thioester bond. The traditional method for detecting palmitoylated proteins is to use radioactive palmitate to metabolically label cultured cells, and detect the degree of isotope labeling by autoradiography. It can be used...

AI Technical Summary

Problems solved by technology

The traditional method for detecting palmitoylated proteins is to label cultured cells with radioactive palmitate and detect the degree of isotope labeling by autoradiography. It can be used to detect palmitoylated modified proteins in living cells, but it has defects such as low sensitivity, lack of methods for direct enrichment and identification of radioactively labeled proteins

In the prior art, there are two main types of methods for protein palmitoylation flux analysis, one of which is a "palmitate-centered" analysis method, that is, using azide- or alkyne-containing palmitate-like Metabolic labeling of cultured cells, through chemoselective linking such as Staudinger ligation or click chemistry, selectively link proteins with azide or alkyne groups to biotin or fluorescent groups for enrichment or detection Proteins modified by palmitoylation; this type of method cannot analyze tissue samples and body fluids, and may be more biased towards proteins with faster palmitate turnover, and the introduction of palmitate analogs may lead to unpredictable biological effects; another major This method is a "cysteine-centered" method, that is, after protein extraction, all free sulfhydryl groups on the protein Cys are completely blocked with iodoacetamide or N-ethylmaleimide, and then hydroxylamine is used to (HA) selectively cleaves the thioester bond between the protein Cys and the fatty acid modification group to generate a new free sulfhydryl group, and then reacts with a suitable reagent such as a reagent with sulfhydryl specific reactivity to convert it into disulfide Linked biotin, and then purified to detect S-palmitoylated modified protein; this method is an indirect detection and purification method, and the false positive rate is relatively high

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