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Lane 1: HeLa, vehicle (DMSO) treated for 4 hours Lane 2: HeLa 1 µM staurpsorine (ab120056), 4 hours Load 20 µg/lane 5% milk/PBST for block and antibody diluent Primary antibodies (2 hours, room temp) All lanes: ab136812 250X Primary Antibodies Cocktail, 1/250 dilution Secondary antibodies (1 hour, room temp) All lanes: ab136812 100X HRP-Conjugated Secondary Antibodies Cocktail, 1/100 dilution
Example of IC50 determination. HeLa cells were treated with a dose titration of Staurosporine for 4 hours in complete media. Cells were cultured and treated in a 96-well cell culture microtiter plate. Lysates were prepared by direct in-well lysis without media removal: 2X Cell Extraction Buffer PTR was added to an equal volume of media and then resulting lysate was used directly in the Cleaved PARP SimpleStepTM ELISA assay. Raw values for triplicate measurements are plotted. The calculated IC50 is 0.77 µM.
Demonstration of Cleaved PARP capture antibody specificity by western blot assay. 20 µg of HeLa extracts that were untreated or treated for 4 hours with 1 µM Staurosporine were analyzed by western blot. The GAPDH blot is included to show the relative loads of each lysate. In the HeLa cell line, Staurosporine treatment is required to detect cleaved PARP protein, as observed in the SimpleStep ELISA.
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