An Alternative Strategy for Pan-acetyl-lysine Antibody Generation.
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.
Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Sun-Yee
SY
Kim
Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Choon Kiat
CK
Sim
Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Qiongyi
Q
Zhang
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, United States of America.
Hui
H
Tang
Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Reinhard
R
Brunmeir
Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Hong
H
Pan
Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Republic of Singapore.
Neerja
N
Karnani
Laboratory of Metabolic Medicine, Singapore Bioimaging Consortium, Agency for Science, Technology and Research, Singapore, Republic of Singapore.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.|Cardiovascular and Metabolic Disorders Program, Duke-NUS Graduate Medical School, Singapore, Republic of Singapore.
Weiping
W
Han
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, United States of America.
Kangling
K
Zhang
Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore, Republic of Singapore.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Republic of Singapore.|Cardiovascular and Metabolic Disorders Program, Duke-NUS Graduate Medical School, Singapore, Republic of Singapore.
Feng
F
Xu
D041721:3T3-L1 Cells; D000107:Acetylation; D020816:Amino Acid Motifs; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D002853:Chromatography, Liquid; D016384:Consensus Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D063990:Gene Ontology; D057809:HEK293 Cells; D006801:Humans; D007118:Immunoassay; D008239:Lysine / Q000378:metabolism*; D008297:Male; D055442:Metabolome; D051379:Mice; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
D000906:Antibodies; D010455:Peptides; D008239:Lysine