Example of Active Caspase 3 standard curve for measurement of extracts prepared from cell pellets and tissue homogenates. Background-subtracted data values (mean +/- SD, n=2) are graphed.
Example of Active Caspase 3 standard curve for measurement of lysates prepared from cells in media (RPMI1640 supplemented with 10% Fetal Bovine Serum) by direct in well lysis. Background-subtracted data values (mean +/- SD, n=2) are graphed.
Titration of Jurkat Cell Extract prepared from pellets of cells treated for 4 hours with 1 µM Staurosporine or vehicle (DMSO) within the working range of the assay. Raw data values (mean +/- SD, n=2) are graphed. Dotted line represents Blank control.
Titration of HeLa Cell Extract prepared from pellets of cells treated for 4 hours with 1 µM Staurosporine or vehicle (DMSO) within the working range of the assay. Raw data values (mean +/- SD, n=2) are graphed. Dotted line represents Blank control.
Example of Staurosporine IC50 determination. Jurkat Cell Lysates corresponding to 2x106 cells/mL or 1x106 cells/mL were prepared by direct in-well lysis (without media removal) from cells grown in RPMI1640 media supplemented with 10% FBS and treated for 4 hours with variable doses of Staurosporine in a 96-well plate. Background-subtracted data values (mean +/SD, n=3) are graphed. IC50 determined from background-subtracted data were 1.1 µM and 0.9 µM using, respectively, lysates of 2x106 cells/mL and 1x106 cells/mL.
Comparison of active caspase 3 concentration in Jurkat Cell Extracts prepared from pellets of cells treated for 4 hours with 1 µM Staurosporine or drug’s vehicle (DMSO) using Active Caspase 3 (Ser29) Human SimpleStep™ ELISA Kit. Background-subtracted data values (mean +/- SD, n=2) of several extract concentrations analyzed (as indicated) are graphed. Note that the Active Caspase 3 is detectable only in cells undergoing apoptosis induced by Staurosporine treatment. This result correlates well with Western blot analysis.
Quantification of Active Caspase 3 concentration in Jurkat Cell Extracts prepared from pellets of cells treated for 4 hours with 1 µM Staurosporine or drug’s vehicle (DMSO) using Active Caspase 3 (Ser29) Human SimpleStep™ ELISA Kit. The concentrations of Active Caspase 3 were interpolated from data values shown in Figure 6 using Active Caspase 3 standard curve, corrected for sample dilution, and graphed in ng of Active Caspase 3 per mg of extract. Note that the Active Caspase 3 is detectable only in cells undergoing apoptosis induced by Staurosporine treatment. This result correlates well with Western blot analysis.
Demonstration of the capture and detector antibodies specificities. Active Caspase 3 protein (ab52314, lane 1, 8 ng/lane) and 20 µg/lane of cell extracts prepared from 4 hours vehicle-treated (lane 2) and 1 µM Staurosporine-treated (lane 3) Jurkat cells were analyzed by Western blotting using the Active Caspase 3 (Ser29) Capture Antibody (A), or the Active Caspase 3 (Ser29) Detector Antibody (B). Note that the Active Caspase 3 (Ser29) Capture Antibody used in this kit specifically detects only the p17 subunit of Active Caspase 3 but not the pro-caspase 3 in Jurkat Cell Extracts.