An affinity labeling of ras p21 protein and its use in the identification of ras p21 in cellular and tissue extracts

• Published in: The Journal of biological chemistry 1987-02, Vol.262 (5), p.2369-2373
• Main Authors: Basu, A., Modak, M.J.
• Format: Article
• Language: English
• Subjects: Affinity Labels - metabolism; Animals; Biological and medical sciences; Cell Extracts - analysis; Cell physiology; Cell transformation and carcinogenesis. Action of oncogenes and antioncogenes; Ethylmaleimide - pharmacology; Fundamental and applied biological sciences. Psychology; Guanosine Triphosphate - metabolism; Kinetics; Liver Neoplasms, Experimental - metabolism; Mice; Molecular and cellular biology; Photochemistry; Protein Conformation; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins p21(ras); Tissue Extracts - analysis; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)61664-3

We have carried out photoaffinity labeling of the ras p21 protein, a ras oncogene product, with [alpha-32P]GTP. Based on our studies, a sensitive, rapid, and specific assay for the detection of multiple forms of ras p21 has been developed. The specificity of this protocol is shown by (a) sensitivity of affinity labeling of ras p21 to known inhibitors of GTP binding and (b) immunoprecipitation of affinity labeled protein with anti-ras p21 serum. Detection and semiquantitation of ras p21 by this method is accomplished in less than 24 h and requires as little as 100,000 cells or about 5 mg of tissue sample from skin tumor, liver, and mammary tumor tissues. Furthermore, using this approach, we were able to detect the selective loss of one species of ras p21 in transplanted Morris hepatoma cells.