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

Proteome Science
Ziqian LiangGang Li

Abstract

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. 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. 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. The specific tMeK antibody we developed is useful...Continue Reading

References

Oct 1, 2004·The Journal of Biological Chemistry·Bettina SargHerbert H Lindner
Jan 18, 2006·Nature Structural & Molecular Biology·Jean-François CoutureRaymond C Trievel
Nov 17, 2006·Nature·Jing HuangShelley L Berger
Mar 22, 2007·Mutation Research·Xiaodong Cheng, Xing Zhang

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Citations

Jan 19, 2016·Journal of Molecular Biology·Qianting ZhaiPaul J Carter
Aug 3, 2014·Biochemical and Biophysical Research Communications·Lelin ZhangMarc R Wilkins

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Methods Mentioned

BETA
immunoprecipitation
ELISA
affinity purification
protein modification
Protein Assay

Software Mentioned

Northern Eclipse imaging

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