A NOVEL TECHNIQUE FOR THE STUDY OF RAS ACTIVITY - ELECTROPORATION OF [ALPHA-P-32]GTP

An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [alpha -P-32]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 degrees C, Ras was immunoprecipitated from cell ex tracts and the Ras-bound GTP/GTP + GDP ratio was determined by thin-la yer chromatography, Contrary to Streptolysin-O permeabilization, the c ells are not affected in any detectable way by the procedure, so that [alpha-P-32]GTP binding and conversion to [alpha-P-32]GDP can be studi ed over a period of time for the measurement of steady-state Ras activ ity, The results show that careful control of electric field intensity results in a great increase in the efficiency and specificity of labe lling compared to the addition of [P-32]orthophosphate to the culture medium, while the GTP/GTP + GDP ratios obtained were essentially the s ame as after in vivo labeling.