A method of detecting protein lysine crotonylation acylation modification and developing affinity reagents

A technique for crotonylation and histone, applied in the field of reagents for detecting protein crotonylation

Active Publication Date: 2013-07-03
JINGJIE PTM BIOLAB HANGZHOU CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although initially found in histones, lysine acetylation

Method used

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  • A method of detecting protein lysine crotonylation acylation modification and developing affinity reagents
  • A method of detecting protein lysine crotonylation acylation modification and developing affinity reagents
  • A method of detecting protein lysine crotonylation acylation modification and developing affinity reagents

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Embodiment 1

[0118] All polypeptides of the present invention are entrusted to a synthetic company to synthesize, and Fmoc-Lysine (crotonylated tonyl)-OH is used to protect amino acids. All chemical reagents and Flag M2 antibody were purchased from Sigma-Aldrich (St. Louis, MO). All chemicals were of the highest purity or analytical grade commercially available. HA antibody was purchased from Roche Diagnostics (Indianapolis, IN). Histones were extracted using previously reported methods (see Shechter, D., Dormann, H.L., Allis, C.D., and Hake, S.B. (2007). Extraction, purification and analysis of histones. Nat Protoc 2, 1445-1457.; Tateishi , K., Okada, Y., Kallin, E.M., and Zhang, Y. (2009). Role of Jhdm2a in regulating metabolic gene expression and obesity resistance. Nature 458, 757-76 1.) From S. cerevisiae S cells, S2 cells, mouse embryonic fibroblast (MEF) cells, Caucasian human embryonic lung fibroblasts (IMR90) and human HeLa cells. 4,4,4,3-D4-Crotonic acid was synthesized from ...

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Abstract

The invention provides an antibody, which specifically binds a histone or histone segment having crotonyllysine residues, a method for detecting protein lysine crotonylation by the antibody specifically binding histone or histone segment having crotonyllysine residues when crotonylation occurs at the histone or histone segment (preferably at the crotonylation site). The invention also provides a fusion indication protein. The core of the fusion indication protein is that: one side of the protein is provided with a fluorescence donor part and one side of the protein is provided with a fluorescence receptor part, and the protein is composed of a histone polypeptide and a crotonylation binding domain. The crotonylation binding domain only specifically binds the histone polypeptide when crotonylation occurs at the histone polypeptide (preferably at the crotonylation site). The invention also provides a method for determining the protein lysine crotonylation level by the fusion indication protein.

Description

technical field [0001] The invention relates to a reagent and a method for detecting protein crotonylation. More specifically, it is related to polypeptides derived from histones or histone fragments containing crotonylation sites, and using these polypeptides to develop reagents and methods for detecting protein crotonylation. Background technique [0002] The molecular anatomy of post-translational modifications (PTMs) that regulate cellular processes and disease progression is one of the main goals of post-genomic biology research. To date, more than 300 post-translational modifications have been discovered. This is an effective way to alter the basic structure and even function of proteins. Modifications on the side chains of lysines demonstrate the enormous complexity of the molecular network. Lysine is an amino acid residue encoded by 15 ribosomes and known to be modifiable. The lysine side chain has two characteristics: rich charge and nucleophilicity, suitable fo...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/08C07K16/18C07K16/06G01N33/68G01N21/64A61K45/00A61P43/00
CPCA61P43/00C07K14/4702
Inventor 程仲毅赵英明
Owner JINGJIE PTM BIOLAB HANGZHOU CO LTD
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