Unexpected biotinylation using ATP-γ-Biotin-LC-PEO-amine as a kinase substrate.

Department of Chemistry and the Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, NY 11794, USA.

Biochemical and biophysical research communications 2013 Mar 8

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Protein phosphorylation is the most widely studied post-translational modification. Reversible protein phosphorylation is implicated in the regulation of a broad range of cellular processes. As such, there is extensive interest in simple and sensitive procedures for the isolation and detection of phosphorylated proteins. Synthetic analogues of ATP, with a biotin linked to the gamma-phosphate of ATP, have been reported to biotinylate kinase substrates in a kinase-catalyzed reaction. This could be an extremely attractive and versatile method for affinity enrichment of phosphorylated proteins. However, as we report here, the commercially available biotin-ATP analogue, ATP-γ-Biotin-LC-PEO-amine, is capable of biotinylating proteins independent of kinase activity. In fact, we demonstrate that this reagent is capable of non-specifically biotinylating any protein. Although the mechanism of biotinylation is not known, this report uncovers a flaw in a commercially available reagent and also highlights the importance of control experiments when developing new biochemical tools to study enzyme activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Citation

Dhruv P Arora, Elizabeth M Boon. Unexpected biotinylation using ATP-γ-Biotin-LC-PEO-amine as a kinase substrate. Biochemical and biophysical research communications. 2013 Mar 8;432(2):287-90