Development of pan-specific antibody against trimethyllysine for protein research.
Liang Z, Wong RP, Li LH, Jiang H, Xiao H, Li G.
Rehabilitation Center of Burns and Plastic Surgery and Medical Research Center, Guangxi Medical University, Nanning, China. info@immunechem.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 - in process]PMCID: PMC2267453