Parallelism experiments were carried out to determine if the recombinant standard accurately determines [pSer326] HSF1 concentrations in biological matrices. Values were obtained using cell lysates from treated and untreated cells serially diluted in the assay buffer and assessed from a standard curve using four parameter logistic curve fitting.The observed values were plotted against the dilution factors. Parallelism of the curves demonstrates that the antigen binding characteristics are similar enough to allow the accurate determination of native analyte levels in diluted samples.

HeLa and 3T3 cells were grown to approximately 80% confluency and subjected to heat shock at 42ºC for 2 hours. Extracts were prepared as described in “Sample Preparation” and the levels of HSF1 were determined in the assay. Induction is expressed as the fold change relative to non heat-shocked control samples.