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SIRT3 and SIRT6 activity assay

ZY Zhi-qiang Yao
XZ Xin Zhang
YZ Yongqi Zhen
XH Xu-Ying He
SZ Shuangmei Zhao
XL Xi-Feng Li
BY Bo Yang
FG Feng Gao
FG Fu-You Guo
LF Leilei Fu
XL Xian-Zhi Liu
CD Chuan-Zhi Duan
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The SIRT3 and SIRT6 activity assay were performed using SIRT3 Activity Assay Kit (Enzo Life Sciences, BML-AK557) and SIRT6 Activity Assay Kit (abcam, ab156068) according to the manufacturer’s instructions. In brief, Fluoro-Substrate Peptide, Fluoro-Deacetylated Peptide and NAD were successively added to SIRT3 and SIRT6 assay buffer followed by incubation with the tested compounds, as well as the recombinant SIRT (5 mg) at 37 °C for 45 min. For SIRT3 assay, stop the assay with 1× Developer II/2 Mm nicotinamide, excitation at 360 nm, emitted light was detected at 460 nm using microtiter plate fluorometer. For SIRT6 assay, excitation at 480 nm, emitted light was detected at 530 nm using microtiter plate fluorometer. The fluorescence intensity of the assay buffer was subtracted from each experimental sample.

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