Kim SY, Sim CK, Zhang Q, Tang H, Brunmeir R, Pan H, Karnani N, Han W, Zhang K, Xu F. “An Alternative Strategy for Pan-acetyl-lysine Antibody Generation”. PLoS One. 2016 Sep 8;11(9):e0162528. doi: 10.1371/journal.pone.0162528.
Lysine acetylation is an important post-translational modification in cell signaling. In acetylome studies, a high-quality pan-acetyl-lysine antibody is key to successful enrichment of acetylated peptides for subsequent mass spectrometry analysis. Here we show an alternative method to generate polyclonal pan-acetyl-lysine antibodies using a synthesized random library of acetylated peptides as the antigen. Our antibodies are tested to be specific for acetyl-lysine peptides/proteins via ELISA and dot blot. When pooled, five of our antibodies show broad reactivity to acetyl-lysine peptides, complementing a commercial antibody in terms of peptide coverage. The consensus sequence of peptides bound by our antibody cocktail differs slightly from that of the commercial antibody. Lastly, our antibodies are tested in a proof-of-concept to analyze the acetylome of HEK293 cells. In total we identified 1557 acetylated peptides from 416 proteins. We thus demonstrated that our antibodies are well-qualified for acetylome studies and can complement existing commercial antibodies.
This work was supported by the intramural funding from the Agency for Science, Technology and Research (A*STAR) of Singapore to FX. Funding for the open-access charge was provided by A*STAR of Singapore. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.