Using the PhosphoTracer phospho-p53 assay, the length of assay incubation time that was required to detect recombinant phospho-p53 was examined. After 15 mins, phospho-p53 concentrations of approximately 2 ng/ml were readily detected, while after 2 hour incubation, the limit of detection was approximately 0.2 ng/ml. Longer incubation times had little effect on overall limit of detection that was observed.

U2OS Cells were seeded at 25K cells/well in a 96 well tissue culture plate. The following day, the media was aspirated and replaced with fresh media containing doxorubicin at various concentrations. The plate was incubated at 37°C for a further 18 hours. The media was removed from the wells, and the cells were lysed with 150 µL/well freshly prepared PhosphoTracer Lysis Mix, with shaking for 10 min at room temp. Lysates (50 µL) were transferred to replicate wells of a PhosphoTracer assay plate and analyzed for either phospho-p53, or Total p53 using the standard PhosphoTracer protocol. Briefly, Antibody Mix specific for either phospho-p53, or Total p53 was added to the lysates, and the plate was incubated for 1 hr at room temp, with shaking. The plate was washed and Substrate Mix was added. The plates were covered in foil and incubated for 10 min with shaking. Signal in the wells was determined using a plate reader.