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We describe a microplate-based assay for NAD-dependent Class III histone deacetylases (also known as SIRTs) that measures the enzyme-catalyzed release of nicotinamide from radiolabeled NAD, using a boronic acid resin to selectively capture the NAD. This method avoids the need for fluorogenic or radiolabeled peptides or separation of the reaction products using solvent extraction. The protocol reported...
Class III histone deacetylases, or sirtuins, are homologous to the Saccharomyces cerevisiae transcriptional regulator SIR2. The class III enzymes are characterized by their dependence on nicotinamide adenine dinucleotide (NAD + ). This cofactor serves as an acetyl-group acceptor in the deacetylation reaction generating O-acetyl-ADP-ribose. Enzymatic activity of sirtuin can be measured in vitro...
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