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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.


Related in: MedlinePlus

Western blot and immunoprecipitation analysis of proteins from human melanoma cells using the anti-tMeK antibody. A. Cells treated with or without TSA were lysed for Western blot (50 μg/lane) using the trimethyllysine-purified antibody, with and without the presence of free tMeK. B. Western blot analysis of the anti-tMeK immunoprecipitated proteins from the crude lysate of the mouse spleen using anti-tMeK HRP conjugates (0.25 μg/ml). The signal was competitively inhibited by the free tMeK (10 μg/ml). C. Western blot analysis of immunopreciptates by IgG control or anti-tMek antibody with H3K4me3, H3K9me3, or H3K27me3 antibodies.
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Figure 3: Western blot and immunoprecipitation analysis of proteins from human melanoma cells using the anti-tMeK antibody. A. Cells treated with or without TSA were lysed for Western blot (50 μg/lane) using the trimethyllysine-purified antibody, with and without the presence of free tMeK. B. Western blot analysis of the anti-tMeK immunoprecipitated proteins from the crude lysate of the mouse spleen using anti-tMeK HRP conjugates (0.25 μg/ml). The signal was competitively inhibited by the free tMeK (10 μg/ml). C. Western blot analysis of immunopreciptates by IgG control or anti-tMek antibody with H3K4me3, H3K9me3, or H3K27me3 antibodies.

Mentions: Figure 3a showed that the anti-tMeK antibody recognized various proteins in the human melanoma cells. Those protein bands recognized by the anti-tMeK antibody could be completely inhibited by the free tMeK suggesting that the antibody is Nε-trimethyllysine-specific and the protein bands recognized by the anti-tMeK are trimethylated.


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)

Western blot and immunoprecipitation analysis of proteins from human melanoma cells using the anti-tMeK antibody. A. Cells treated with or without TSA were lysed for Western blot (50 μg/lane) using the trimethyllysine-purified antibody, with and without the presence of free tMeK. B. Western blot analysis of the anti-tMeK immunoprecipitated proteins from the crude lysate of the mouse spleen using anti-tMeK HRP conjugates (0.25 μg/ml). The signal was competitively inhibited by the free tMeK (10 μg/ml). C. Western blot analysis of immunopreciptates by IgG control or anti-tMek antibody with H3K4me3, H3K9me3, or H3K27me3 antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Western blot and immunoprecipitation analysis of proteins from human melanoma cells using the anti-tMeK antibody. A. Cells treated with or without TSA were lysed for Western blot (50 μg/lane) using the trimethyllysine-purified antibody, with and without the presence of free tMeK. B. Western blot analysis of the anti-tMeK immunoprecipitated proteins from the crude lysate of the mouse spleen using anti-tMeK HRP conjugates (0.25 μg/ml). The signal was competitively inhibited by the free tMeK (10 μg/ml). C. Western blot analysis of immunopreciptates by IgG control or anti-tMek antibody with H3K4me3, H3K9me3, or H3K27me3 antibodies.
Mentions: Figure 3a showed that the anti-tMeK antibody recognized various proteins in the human melanoma cells. Those protein bands recognized by the anti-tMeK antibody could be completely inhibited by the free tMeK suggesting that the antibody is Nε-trimethyllysine-specific and the protein bands recognized by the anti-tMeK are trimethylated.

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.


Related in: MedlinePlus