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Generic methylation and dimethylation antibody and preparation method thereof

A dimethylation and methylation technology, applied in the field of biomedicine, can solve problems such as the lack of antibodies that specifically recognize trimethylated proteins

Active Publication Date: 2016-06-29
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a novel pan-mono- and di-methylated antibody and its preparation method to solve the current problem that there is no antibody that specifically recognizes tri-methylated proteins

Method used

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  • Generic methylation and dimethylation antibody and preparation method thereof
  • Generic methylation and dimethylation antibody and preparation method thereof
  • Generic methylation and dimethylation antibody and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] One: Preparation of bovine serum albumin BSA that simulates cysteine ​​into monomethylated lysine (Kc[me1])

[0029] 1: Dissolve 10mg BSA bovine serum albumin BSA in 900ul alkylation reagent (1M 4-hydroxyethylpiperazineethanesulfonic acid, pH7.8; 4M guanidine hydrochloride 10mML-methionine);

[0030] 2: Add 20ul of 1M dithiothreitol (DTT), and react at 37°C for 1 hour;

[0031] 3: Add 100ul of 1M (2-chloroethyl)-methylammonium chloride, and carry out alkylation reaction at room temperature for 4 hours;

[0032] 4: Add 10ul1M dithiothreitol (DTT) and react at room temperature for 10 hours;

[0033] 5: Add 50ul of 14.2M mercaptoethanol (BME) to terminate the reaction to obtain simulated monomethylated bovine serum albumin BSA (Kc[me1]).

[0034] Two: Preparation of bovine serum albumin BSA that simulates cysteine ​​into dimethylated lysine (Kc[me2])

[0035] 1: Dissolve 10mg BSA bovine serum albumin BSA into 930ul alkylation reagent (1M 4-hydroxyethylpiperazineethanesu...

Embodiment 2

[0044] 1: Preparation of hemocyanin KLH that simulates cysteine ​​into monomethylated lysine (Kc[me1])

[0045] 1: Weigh 10mg hemocyanin KLH and dissolve it into 900ul alkylation reagent (1M 4-hydroxyethylpiperazineethanesulfonic acid, pH7.8; 4M guanidine hydrochloride 10mML-methionine);

[0046] 2: Add 20ul of 1M dithiothreitol (DTT), and react at 37°C for 1 hour;

[0047] 3: Add 100ul of 1M (2-chloroethyl)-methylammonium chloride, and carry out alkylation reaction at room temperature for 4 hours;

[0048] 4: Add 10ul1M dithiothreitol (DTT) and react at room temperature for 10 hours;

[0049]5: Add 50 ul of 14.2 M mercaptoethanol (BME) to terminate the reaction to obtain hemocyanin KLH (Kc[me1]) which simulates monomethylation modification.

[0050] Two: Preparation of hemocyanin KLH that simulates cysteine ​​into dimethylated lysine (Kc[me2])

[0051] 1: Weigh 10mg hemocyanin KLH and dissolve it in 930ul alkylation reagent (1M 4-hydroxyethylpiperazineethanesulfonic acid, ...

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Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses a generic methylation and dimethylation antibody and a preparation method thereof. Polypeptide coupling carrier protein such as bovine serum albumin or ovalbumin or keyhole limpet hemocyanin or bovine thyroglobulin directly serves as an immunizing antigen premise substrate, and cysteine in the carrier protein is subjected to alkylation modification to be simulated into methylation modified lysine (Kc[me1]) and dimethylation modified lysine (Kc[me2]) respectively through alkylating reagents of (2-chloroethyl)-methylammonium chloride and (2-chloroethyl)-dimethylammonium chloride by means of a chemical modification method; a mixture of Kc[me1] and Kc[me2] serves as an immunizing antigen to immunize rabbits, and an antibody capable of specially recognizing methylation protein and dimethylation protein is obtained. The generic methylation and dimethylation antibody and multiple histone polypeptides subjected to methylation modification are used for conducting immunoprecipitation experiments and conducting western blot detection. It is shown through results that the obtained generic methylation and dimethylation antibody can specially recognize the histone polypeptides subjected to methylation and dimethylation modification but almost cannot recognize histone polypeptides which are not subjected to methylation modification or subjected to trimethylation modification.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a pan-monomethylated antibody and a preparation method thereof. Background technique [0002] Lysine can undergo methylation modification, including one, two, and three methylation modifications. Past studies have paid more attention to the lysine methylation modification of histones. However, recently more and more evidences have shown that lysine on non-histone proteins can also undergo methylation modification, and these methylation modifications also play an important role in the occurrence and development of diseases, so the identification of non-histone proteins The methylation modification of lysine on lysine also appears to be crucial, which requires effective methods to enrich methylated proteins for mass spectrometry identification, the most important method of which is enrichment with antibodies. [0003] At present, there is no pan-dimethylation antib...

Claims

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Application Information

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IPC IPC(8): C07K16/06
CPCC07K16/06C07K2317/10
Inventor 蓝斐沈宏杰徐文绮
Owner FUDAN UNIV
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