The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16). Plate-based assays such as this can be imaged on the Odyssey® CLx or Odyssey Sa Infrared Imaging System.
So you are hopefully ready to give this technique a try. When you do, it is important to assess the overall quality and reliability of the assay during In-Cell Western (ICW) assay optimization. The Z’-factor statistic provides a way to evaluate whether or not assay conditions (reagents, protocols, instrumentation, kinetics, and other conditions not directly related to the test compounds) are optimized. Z’-factor, introduced by Zhang et al., is a dimensionless value that represents both the variability and the dynamic range between two sets of sample control data.
Z’-factor experiments are performed on one or more ICW assay plates containing replicate wells designated for background subtraction, negative control samples, and positive control samples. Typically, negative control wells are those in which the cells receive an the appropriate treatment so as to elicit the lowest desired percent response (usually untreated cells); positive control wells are those in which the cells receive an appropriate treatment so as to elicit the maximum desired percent response; background wells are treated the same as the negative control wells, except primary antibody incubation is excluded.