In-Cell Westerns use a two-color ratiometric approach for precise, quantitative analysis of cell signaling with good reproducibility.

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Reproducibility of the ICW assay*. IC50 values were determined for four experimental compounds that inhibit PDGF-induced phosphorylation of Akt in NIH 3T3 cells. At each drug concentration, the integrated fluorescence intensity for phospho-Akt staining was determined. For each data point, eight replicates were performed; the mean and standard deviation are shown. Each experiment was repeated five times (plates 1-5) and IC50 values for each plate are shown. Each compound gave IC50 values that were indistinguishable across the five replicates.