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Cell lysates were added to individual wells of a 96-well plate so that active Cdc42-GTP would be immobilized by immunoaffinity to the bottom surface of each well. Washing steps removed inactive Cdc42-GDP so that Cdc42-GTP remaining in each well could be detected using an anti-Cdc42 antibody in combination with a secondary antibody conjugated to horseradish peroxidase (HRP). Oxidation of an added substrate was monitored so that the reaction product could be quantified using a BioTek Synergy HTX Multi-Mode Plate Reader.
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