title: A novel technique for the study of Ras activity: electroporation of [alpha-32P]GTP.
abstract: An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [alpha-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 degrees 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 [alpha-32P]GTP binding and conversion to [alpha-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.
authors.affiliation: Department of Microbiology, Queen's University, Kingston, Ontario, Canada.
authors.forename: H L
authors.initials: HL
authors.lastname: Brownell
authors.forename: K L
authors.initials: KL
authors.lastname: Firth
authors.forename: K
authors.initials: K
authors.lastname: Kawauchi
authors.forename: T
authors.initials: T
authors.lastname: Delovitch
authors.forename: L
authors.initials: L
authors.lastname: Raptis
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D018274:Electroporation; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D051379:Mice; D015689:Oncogene Protein p21(ras) / Q000378:metabolism*; D010761:Phosphorus Radioisotopes
chemical_list: D010761:Phosphorus Radioisotopes; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D015689:Oncogene Protein p21(ras)