Abstract
The histone deacetylase known as sirtuin 6 (SIRT6) deacetylates both histone and non-histone proteins but has low deacetylase activity in vitro. Here, we present a protocol to monitor SIRT6-mediated deacetylation of long-chain acyl-CoA synthase 5 in the presence of palmitic acid. We describe the purification of His-SIRT6 and a Flag-tagged substrate. We then detail a deacetylation assay protocol that can be widely applied to study other SIRT6-mediated deacetylation events and the effect of SIRT6 mutations on its activity. For complete details on the use and execution of this protocol, please refer to Hou et al. (2022).1.