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In-Cell Westerns
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In-Cell Western Immunofluorescent Assays

Application Examples

In-Cell Westerns offer broad application to the analysis of protein signaling pathways, reliable protein quantification, and cell-based determinations of IC₅₀ concentrations for lead optimization. In-Cell Westerns are also a powerful tool in the study of the effects of drug components on multiple points within one or more signaling pathways.

Other traditional protein assay methods such as Western blotting are cumbersome and labor intensive. High content screening methods employ very expensive complex instrumentation. In-Cell Westerns offer a practical alternative for medium to high throughput analysis.

Phosphorylation of ERK in Response to Pathway Stimulation

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A431 cells were stimulated with serial dilutions of EGF to optimize activation of ERK1/2*.

A) Detection of ERK phosphorylation. These images show a portion of a 96-well plate. The top panel is a composite image showing the fluorescence in both the 700 nm and 800 nm detection channels. Duplicate rows of microplate wells are shown. The middle panel shows detection of total ERK protein regardless of phosphorylation status. The bottom panel shows detection of increasing amounts of phospho-ERK as a function of increasing EGF stimulation.

B) Quantification of fluorescence. Phospho-ERK signal has been normalized using the total ERK signal from each well, to correct for well-to-well variation in cell number. This experiment shows a greater than 16-fold increase in ERK phosphorylation compared to the resting state.