A Novel Technique for the Study of Ras Activity: Electroporation of [α- 32 P]GTP

Abstract

An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [α-³²P]GTP was introduced by in situ electroporation into mouse C3H10T1/2 fibroblasts or their ras ^val12-transformed derivatives. After a 3-hr incubation at 37°C, Ras was immunoprecipitated from cell extracts and the Ras-bound GTP/GTP + GDP ratio was determined by thin-layer chromatography. Contrary to Streptolysin-O permeabilization, the cells are not affected in any detectable way by the procedure, so that [α-³²P]GTP binding and conversion to [α-³²P]GDP can be studied over a period of time for the measurement of steady-state Ras activity. The results show that careful control of electric field intensity results in a great increase in the efficiency and specificity of labelling compared to the addition of [³²P]orthophosphate to the culture medium, while the GTP/GTP + GDP ratios obtained were essentially the same as after in vivo labeling.