- Home
- Protocols
-
Request a Protocol
Ask a
question
Serum starved cells were stimulated with 10% FBS (final concentration) for 1–12 min to activate RAS. Cells were washed with cold PBS, lysed with TX100 buffer (25 mM TRIS pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton) supplemented with protease and phosphatase inhibitors (Roche) and incubated for 1 h at 4 °C with rotation. An aliquot was removed for analysis of total RAS in the whole cell lysate and the remainder incubated with 10ug GST-tagged Raf-binding domain (RBD, gift from Ikura lab). Complexes were immunoprecipitated with magnetic glutathione-agarose beads (Sigma Aldrich). IPs were washed with TX100 buffer and RAS-GTP eluted by boiling in 2X sample buffer.
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Post a Question
