Example of positive control sample standard curve. A dilution series of extract in 1X Incubation Buffer in the working range of the assay. The extract was prepared with Hek293T cells grown in High Glucose DMEM supplemented with 10% FCS
Hek293T extracts were left untreated (control), treated with heat only at 34˚C (Mock) or treated with 1:100 dilution of λ Ppase at 34˚C. Samples were loaded at 750 µg/mL on the plate and measured following the kit’s protocol. Treatment of Hek293T extracts with λ Ppase decreases the signal to background levels.
The detector antibody used in this kit specifically detects the phosphorylated SIRT1 as determined by Western blotting. Hek293T extracts were left untreated (lane 1), treated with heat only (lane 2) or treated with 1:100 dilution of λ Ppase at 34˚C (lane 3). Samples were then diluted in SDS-PAGE buffer and loaded at 30 µg/well. Membranes were blocked with the kit’s blocking reagent at 1X concentration and incubated with either the detector antibody against SIRT1 phospho S47 (A) or the capture antibody against total SIRT1 (B) and label with secondary antibodies conjugated to HRP. λ Ppase completely dephosphorylates SIRT1 without affecting the protein levels.