Quantification of absolute Ras-GDP/GTP levels by HPLC separation of Ras-bound [(32)P]-labelled nucleotides.

Ras guanine nucleotide binding protein (GTPase) activation is a widely assessed readout in cell biological studies. We describe an improved approach for the quantitative analysis of total GDP and GTP bound to Ras. The present method involves HPLC separation and online detection/quantitation of Ras-bound [(32)P]-labelled GDP/GTP. As compared to standard approaches that are time consuming and/or provide only semi-quantitative data, this technique allows the rapid processing of large numbers of samples for the quantitative determination of Ras-bound GDP and GTP.