Development of pan-specific antibody against trimethyllysine for protein research

• Main Authors: Xiao Hao, Jiang Hesheng, Li Lin Hong, Wong Ronald PC, Liang Ziqian, Li Gang
• Format: Article
• Language: English
• Published: BMC 2008-01-01
• Series: Proteome Science
• Online Access: http://www.proteomesci.com/content/6/1/2

<p>Abstract</p> <p>Background</p> <p>Trimethylation of the N<b>ε</b>-lysine residues in a protein is one of the most important events of posttranslational modifications. Simple methods for rapid detection and isolation of the N<b>ε</b>-trimethylated protein species are needed. This report introduces a novel method to prepare the affinity purified antibody specific for the N<b>ε</b>-trimethylated lysine (tMeK). The applications of the purified antibody are also reported in this paper.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>The ELISA results indicated that the antibody reacted only to tMeK but not to mono- and dimethyllysine. Western-blot results showed that the N<b>ε</b>-trimethylated proteins were detected in both animal tissue and cultured cells and that the antibody signal could be competitively inhibited with free tMeK.</p> <p>Conclusion</p> <p>The specific tMeK antibody we developed is useful for one-step isolation of proteins with N<b>ε</b>-trimethyllysine residues and also for the detection, identification and localization of proteins with trimethyllysine residues in the cells.</p>