Example positive control sample standard curve. A dilution series of extract in Incubation Buffer in the working range of the assay. The extract was prepared from COS7 cells serum-starved overnight and then treated with 200 nM Phorbol 12-myristate 13-acetate (PMA) for one hour.
Example experimental analysis of drug treatment of HeLa cells. Cells were treated with Ser/Thr phosphatase inhibitor Calyculin A, Phorbol 12-myristate 13-acetate (PMA) or drug’s vehicle, as indicated. The Relative levels of BAD Phospho S75 in HeLa cells interpolated from standard curve of vehicle-treated HeLa cells are shown.
Example experimental analysis of drug treatment of COS7 cells. Cells were treated with Ser/Thr phosphatase inhibitor Calyculin A, Phorbol 12-myristate 13-acetate (PMA) or drug’s vehicle, as indicated. Relative levels of BAD Phospho S75 in COS7 cells interpolated from standard curve of vehicle-treated COS7 cells are shown.
The BAD Phospho S75 ELISA specifically measures the phosphorylated Serine. Extracts of COS7 cells (induced with PMA) were treated with increasing concentrations of λ protein phosphatase or left untreated (Contr), and relative BAD phospho Ser75 were determined using this kit. Dilutions of extracts of COS7 cells treated with PMA were used to construct the standard curve.
The detector antibody used in this kit specifically detects the phosphorylated BAD as determined by Western blotting. A and B: Extracts of COS7 cells (induced with PMA) were treated with increasing concentrations of λ protein phosphatase (lane 3, 400x diluted; lane 4, 100x diluted; lane 5, 25x diluted) or left untreated (lane 2). Samples were analyzed by Western blotting using the BAD Phospho S75 Detector Antibody (A) or total BAD protein antibody (B). C and D: Extracts of HeLa cells treated with vehicle (lane 2) or Calyculin (lane 3) and extracts of Cos7 cells treated with vehicle (lane 4) or PMA (lane 5) were analyzed by Western blotting using the BAD Phospho S75 Detector Antibody (C) or total BAD protein antibody (D). Note slower mobility of phosphorylated BAD in COS7 cells compared to dephosphorylated BAD.