Methodology

Development of pan-specific antibody against trimethyllysine for protein research

Ziqian Liang^{* 1} , Ronald PC Wong^{* 2} , Lin Hong Li¹ , Hesheng Jiang¹ , Hao Xiao¹ and Gang Li²

¹Rehabilitation Center of Burns and Plastic Surgery and Medical Research Center, Guangxi Medical University, Nanning, China

²Department of Dermatology and Skin Science, Jack Bell Research Centre, Vancouver Coastal Health Research Institute, University of British Columbia, Vancouver, British Columbia, Canada

author email corresponding author email* Contributed equally

Proteome Science 2008, 6:2doi:10.1186/1477-5956-6-2

Published:	22 January 2008

Abstract

Background

Trimethylation of the Nε-lysine residues in a protein is one of the most important events of posttranslational modifications. Simple methods for rapid detection and isolation of the Nε-trimethylated protein species are needed. This report introduces a novel method to prepare the affinity purified antibody specific for the Nε-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 Nε-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 Nε-trimethyllysine residues and also for the detection, identification and localization of proteins with trimethyllysine residues in the cells.