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Sirtuin Activity Assay

HS Hong Song
LT Loren P. Thompson
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SIRT3 enzyme activity of mitochondrial fractions was measured using the SIRT3 Activity Assay Kit (Fluorometric, Abcam, Cambridge, MA) per the manufacturer’s protocol. Briefly, 50μg of mitochondrial protein was added into individual microplate wells containing a designated amount of fluorescence-labeled acetylated substrate peptide, nicotinamide adenine dinucleotide (NAD), and the developer. Fluorescence intensity was measured (excitation at 350 nm/emission at 450 nm) for 30 min at 1-min intervals by the microplate reader (BioTek, Winooski, VT). Enzyme activity rate was calculated as ΔOD/min.

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