Development of pan-specific antibody against trimethyllysine for protein research

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 CH(3)I 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.