Figure 1. Comparison of permeabilization methods for detection of phospho-Stat3: Z′ factor analysis. A) Cells were fixed with formaldehyde and permeabilized with Triton® X-100. Positive and negative controls overlapped, indicating a poor assay for HTP screening (Z′ = -0.1). B) Cells were fixed and permeabilized with methanol. Wide separation was observed between positive and negative controls, indicating a robust assay for screening (Z′ = 0.7).
Z′ factor is a statistic that measures the reproducibility of an assay for high-throughput screening.
Z′-factor analysis was used to compare Triton X-100 and methanol permeabilization methods for ICW detection of phospho-Stat3 (Fig. 1). EGF-treated and untreated A-431 cells were stained in two independent experiments. Rabbit monoclonal phospho-Stat3 antibody was from Cell Signaling Technology (#9145).