Fluor de lys sirt1 fluorometric drug discovery assay kit

NAD⁺ from brain punches was extracted and measured using the EnzyChrom NAD⁺/NADH Assay Kit (BioAssay Systems, #E2ND-100) according to the manufacturer’s instructions. NAD⁺ levels were normalized to protein content determined by the Lowry method. SIRT1 enzymatic activity was measured using the Fluor de Lys SIRT1 fluorometric drug discovery assay kit (Enzo Life Sciences Inc., #BML-AK555-0001). Briefly, NAc punches were mechanically homogenized with a pellet pestle in lysis buffer [20 mM Hepes (pH 7.4), 2 mM EGTA (pH 8), 1 mM DTT, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1× Mini cOmplete Protease Inhibitor Cocktail], rotated end-over-end for 1 hour at 4°C, and sonicated. Next, 25 μg of protein (diluted in a total volume of 35 μl of assay buffer) was incubated with substrate solution (15 μl) for 45 min at room temperature with shaking. Last, developing solution (50 μl) was added and incubated for 30 min at 37°C. Fluorescence was measured with a microplate reader (excitation, 360 nm; emission, 465 nm; SaFire 2, Tecan).