NAD+ and NADH were then extracted with the extraction buffer provided in the assay kit, mixed with assay buffer, and absorbance-read at 565 nm.
The pellets were then re-suspended with 0.2 mL PBS buffer (pH 7.4) and 0.2 mL NAD + /NADH or NADP + /NADPH lysis buffer provided in the assay kits.
After stimulation with LTD4, cells were washed in PBS and lysed using 250 μl well 1 of the DLR passive lysis buffer provided in the assay.
The initial screens with all 40 compounds contained 5 μl Sirtuin solution (Sirt2/3 0.1 mg/ml stock concentration; Sirt5/6 1 mg/ml), 1 μl of compound stock (resulting in a 100 μM end concentration), 15 μl FdL substrate, and assay buffer (supplied in the assay kit) to a total volume of 50 μl.
Unlike conventional slab gel electrophoresis, submerging the thin fsPAG structure in buffer confounds the assay in three ways.
After the 48 hours of transfections, cellular extracts were collected using the lysis buffer in the dual-luciferase assay (Promega).
SDS-PAGE analysis was performed and the protein bands were stained with Coomassie blue and then transferred to a nitrocellulose membrane by electrotransfer in Towbin's transfer buffer in the western blot assay.
To test H2 production and consumption, cells were grown to stationary phase (16 18 h, 37°C) in LBK buffered at the assay pH in closed screw-cap tubes rotating slowly (8 rpm) at 37°C.
The running buffer used in these experiments mimicked the high salt buffer used in the ATPase assays, buffer D (25 mM HEPES, pH 7.5, 400 mM KCl, 5 mM MgCl2, and 10% DMSO) with 25 µM of 4-OHT or raloxifene.
Following treatment, the cells were trypsinized, washed in assay buffer that was supplied in the kit, and re-suspended in the assay buffer.
