Test method of palmitoylated modified protein based on specific antibody

A palmitoylation and specific antibody technology, applied in the field of biochemical analysis, can solve the problems of lack of direct enrichment and identification, high false positive rate, complicated operation, etc., to achieve simple and easy operability, low false positive rate, specific strong effect

Inactive Publication Date: 2016-11-23
FUDAN UNIV
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AI Technical Summary

Problems solved by technology

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 to detect palmitoylated proteins in living cells, but there are 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 can

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  • Test method of palmitoylated modified protein based on specific antibody
  • Test method of palmitoylated modified protein based on specific antibody
  • Test method of palmitoylated modified protein based on specific antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1 Preparation of immune antigen and control antigen

[0029] The peptides containing palmitoylation modification and their control peptides were synthesized by methods known in the art, and the peptides were coupled to the carrier protein. In this example, Shanghai Ruixing Biotechnology Co., Ltd. completed the synthesis of the peptides Coupling with protein: Synthesize 5 mg of short peptide containing only two cysteines without palmitoylation modification (indicated by C-C) and 9 mg of short peptide containing only two cysteines with palmitoylation modification ( Denote by C-C(pal)), and couple C-C and C-C(pal) to bovine serum albumin BSA, denote as CC-BSA and C(pal)-BSA respectively, and couple C-C(pal) to mcKLH Above, represented by C(pal)-KLH.

Embodiment 2

[0030] Example 2 Preparation of protein-modified pan-antibody serum by antigen immunization

[0031] The prepared C(pal)-KLH immunization experiment rabbits were divided into three immunizations, the first immunization, 200ug / rabbit, the second and third immunization 100ug / rabbit, after the third immunization, routine carotid artery bloodletting, antiserum Store at -20°C after collection.

Embodiment 3

[0032] Example 3 Identification of Antiserum Specificity Using Competitive ELISA

[0033]1) Coating synthetic peptides and proteins C(pal)-KLH, C(pal)-BSA and CC-BSA as antigens at a concentration of 2 μg / ml, dilute the purified antiserum with blocking solution (2 times) (1: 100), carry out ELISA detection, compare the OD value after C(pal)-KLH, C(pal)-BSA and CC-BSA react with antiserum, judge the effect of antiserum, the result shows (as shown in Table 1) , when the antigen is the positive control mcKLH-C-C (pal), there is an obvious immune response; when BSA-C-C (pal) is used as the antigen, there is also a reaction, indicating that the anti-palmitoylation modified antiserum is effective, and its titer is relatively Low; 2) Reduce the dilution ratio of antiserum, dilute the purified antiserum (1:3 start) with blocking solution (2 times), and perform ELISA detection, compare C(pal)-KLH, C(pal) -BSA and CC-BSA reacted with the OD value of the antiserum to judge the effect of...

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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...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/574G01N27/62
CPCG01N33/57484G01N33/6848G01N2440/10
Inventor 陆豪杰方彩云张晓勤
Owner FUDAN UNIV
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