A novel technique for the study of Ras activity: electroporation of [alpha-32P]GTP.
DNA Cell Biol
; 16(1): 103-10, 1997 Jan.
Article
em En
| MEDLINE
| ID: mdl-9022049
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.
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Base de dados:
MEDLINE
Assunto principal:
Proteína Oncogênica p21(ras)
/
Guanosina Trifosfato
Limite:
Animals
Idioma:
En
Ano de publicação:
1997
Tipo de documento:
Article