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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′ is calculated by running a large number of positive and negative controls, and determining how much separation there is between positive and negative controls.
  • If they overlap, or nearly overlap (Z′ < 0.5), due to large amounts of variation within the controls, the assay is useless for screening.
  • An excellent assay exhibits good separation between positive and negative controls (Z′ > 0.5).1-3


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).

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