Pan Ras Monoclonal Antibody (Ras10)

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	-Mouse (1:5000)

Protocol tips
-Mouse (1:5000)

Publication protocol

CRAF expression from both yeast and cell lines was checked by using radioimmune precipitation assay buffer (G-Bioscience, St. Louis, MO) supplemented with a mixture of protease inhibitors (ThermoFisher Scientific), phosphatase inhibitor (ThermoFisher Scientific), and 10 mM PMSF (Sigma). Lysates were cleared by centrifugation at 13,500 rpm for 15 min at 4 °C. For immunoprecipitation, cell lysis was performed in IP buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.l % Nonidet P-40, protease and phosphatase inhibitors, PMSF). For the IP reaction, 800–1000 μg of lysate was incubated with either anti-FLAG or anti-CRAF antibody overnight at 4 °C. 25 μl of protein A/G-Sepharose beads (Calbiochem) was added and incubated for 2 h at 4 °C. The immunocomplex was washed with IP dilution buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.l % Nonidet P-40, protease and phosphatase inhibitors, PMSF) three times, and 20 μl of 2× sample buffer (0.1 M Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 0.2 M β-mercaptoethanol, 0.1% bromphenol blue) was added and boiled for 5 min at 95 °C. The cell lysate or immunocomplex was separated by 10% SDS-PAGE, transferred to membrane, and immunoblotted with respective antibodies.

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