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

Liang Z, Wong RP, Li LH, Jiang H, Xiao H, Li G - Proteome Sci (2008)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rehabilitation Center of Burns and Plastic Surgery, Guangxi Medical University, Nanning, China. liangzqian@yahoo.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.

No MeSH data available.


Generation of anti-trimethyllysine antibody. A. The reaction scheme for chemical synthesis of KLH with trimethyllysine residues. B. The scheme for strategic affinity purification of the trimethyllysine-specific antibody. The antibody in serum cross-reacted to monomethyllysine and dimethyllysine is removed by the mMeK/dMeK affinity column. The remaining trimethyllysine-specific antibody in the serum is further purified using tMeK affinity column.
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Figure 1: Generation of anti-trimethyllysine antibody. A. The reaction scheme for chemical synthesis of KLH with trimethyllysine residues. B. The scheme for strategic affinity purification of the trimethyllysine-specific antibody. The antibody in serum cross-reacted to monomethyllysine and dimethyllysine is removed by the mMeK/dMeK affinity column. The remaining trimethyllysine-specific antibody in the serum is further purified using tMeK affinity column.

Mentions: Ideally, a trimethyllysine-specific antibody (anti-tMeK) should be highly specific and has strong affinity to trimethyllysine (tMeK) but not to monomethyllysine (mMeK) and dimethyllysine (dMeK). The rationale of developing such an antibody is to use a synthetic trimethylated protein, in which, the Nε-amino group of the lysine side chain was trimethylated, as an immunogen. Methylation of the ε-amine groups of a protein using iodomethane (CH3I) generates mono-, di- and trimethylated lysine residues in a protein (Figure 1a). The yield for trimethylated species was increased by performing the reaction at higher pH and in prolonged reaction time (see Materials and Methods). The highly immunogenic protein, KLH, was used for the methylation reaction. The dialyzed methylated KLH was immunized to the rabbit to generate anti-methyllysine antibodies. After 60 days from initial immunization, immune serum was prepared and ELISA test was carried out to test the titer. The serum has 50% maximum OD titer at 1:50,000 to tMeK-BSA conjugates, approximately 1:2,000 and 1:10,000 to mMeK-BSA and dMeK-BSA conjugates respectively (data not shown). The methylated KLH was synthesized while the mMeK, dMeK and tMeK used in ELISA were purchased commercially. The primary ELSIA results indicated that the anti-methylated KLH immune serum cross-reacted with each form of the methylated lysine peptides.


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

Liang Z, Wong RP, Li LH, Jiang H, Xiao H, Li G - Proteome Sci (2008)

Generation of anti-trimethyllysine antibody. A. The reaction scheme for chemical synthesis of KLH with trimethyllysine residues. B. The scheme for strategic affinity purification of the trimethyllysine-specific antibody. The antibody in serum cross-reacted to monomethyllysine and dimethyllysine is removed by the mMeK/dMeK affinity column. The remaining trimethyllysine-specific antibody in the serum is further purified using tMeK affinity column.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267453&req=5

Figure 1: Generation of anti-trimethyllysine antibody. A. The reaction scheme for chemical synthesis of KLH with trimethyllysine residues. B. The scheme for strategic affinity purification of the trimethyllysine-specific antibody. The antibody in serum cross-reacted to monomethyllysine and dimethyllysine is removed by the mMeK/dMeK affinity column. The remaining trimethyllysine-specific antibody in the serum is further purified using tMeK affinity column.
Mentions: Ideally, a trimethyllysine-specific antibody (anti-tMeK) should be highly specific and has strong affinity to trimethyllysine (tMeK) but not to monomethyllysine (mMeK) and dimethyllysine (dMeK). The rationale of developing such an antibody is to use a synthetic trimethylated protein, in which, the Nε-amino group of the lysine side chain was trimethylated, as an immunogen. Methylation of the ε-amine groups of a protein using iodomethane (CH3I) generates mono-, di- and trimethylated lysine residues in a protein (Figure 1a). The yield for trimethylated species was increased by performing the reaction at higher pH and in prolonged reaction time (see Materials and Methods). The highly immunogenic protein, KLH, was used for the methylation reaction. The dialyzed methylated KLH was immunized to the rabbit to generate anti-methyllysine antibodies. After 60 days from initial immunization, immune serum was prepared and ELISA test was carried out to test the titer. The serum has 50% maximum OD titer at 1:50,000 to tMeK-BSA conjugates, approximately 1:2,000 and 1:10,000 to mMeK-BSA and dMeK-BSA conjugates respectively (data not shown). The methylated KLH was synthesized while the mMeK, dMeK and tMeK used in ELISA were purchased commercially. The primary ELSIA results indicated that the anti-methylated KLH immune serum cross-reacted with each form of the methylated lysine peptides.

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rehabilitation Center of Burns and Plastic Surgery, Guangxi Medical University, Nanning, China. liangzqian@yahoo.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.

No MeSH data available.