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Proteome Sci. 2008 Jan 22;6:2. doi: 10.1186/1477-5956-6-2.

Development of pan-specific antibody against trimethyllysine for protein research.

Author information

  • 1Rehabilitation Center of Burns and Plastic Surgery, Guangxi Medical University, Nanning, China. liangzqian@yahoo.com

Abstract

BACKGROUND:

Trimethylation of the Nepsilon-lysine residues in a protein is one of the most important events of posttranslational modifications. Simple methods for rapid detection and isolation of the Nepsilon-trimethylated protein species are needed. This report introduces a novel method to prepare the affinity purified antibody specific for the Nepsilon-trimethylated lysine (tMeK). The applications of the purified antibody are also reported in this paper.

METHODS:

We generated the methylated keyhole limpet heomocyanin (KLH) under controlled chemical methylation reaction using CH3I and used it as an immunogen to raise anti-methylated lysine antibodies. The tMeK specific antibody was selectively isolated using a two-step affinity chromatography in which the mMeK/dMeK specific antibodies were removed and the tMeK specific antibody was captured. Finally, the eluted anti-tMeK antibody was characterized.

RESULTS:

The ELISA results indicated that the antibody reacted only to tMeK but not to mono- and dimethyllysine. Western-blot results showed that the Nepsilon-trimethylated proteins were detected in both animal tissue and cultured cells and that the antibody signal could be competitively inhibited with free tMeK.

CONCLUSION:

The specific tMeK antibody we developed is useful for one-step isolation of proteins with Nepsilon-trimethyllysine residues and also for the detection, identification and localization of proteins with trimethyllysine residues in the cells.

PMID:
18208619
[PubMed]
PMCID:
PMC2267453
Free PMC Article
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