GTP gamma S

Question 1: Can binding of GTP&gamma, S to Ras, Arf, and Rho family GTPases be detected with effector proteins conjugated to Sepharose beads?

Answer 1: Yes, GTP&gamma, S can be used to create activated Ras, Arf, and Rho family GTPases which can be pulled down with Sepharose beads conjugated to an effector protein specific for the GTPase of interest. The GTP&gamma, S should give greater than five-fold increase in activated small G-protein when compared to a GDP bound protein.

Question 2: What percentage of GTPase in an extract will bind to the GTP&gamma, S and so constitute an activated small G-protein?

Answer 2: Approximately 5-10% of the total population of Ras, Arf, or Rho family GTPase will bind to the GTP&gamma, S, creating an activated GTPase protein.