Can you please give advise which of the reagents - GMP-PNP or GTP-gamma-S - is a better choice for activation of Rho family GTPases.
Also, can you recommend the best way of activating Rho GTPase in cells to ensure that they stay active upon cell lysis. I am thinking of calpeptin and GTP-gamma-S in the lysis buffer. Any other suggestions?
Many thanks.
Check this out: J Cell Sci. 1998 Jun;111 ( Pt 11):1583-94. "GTPgS-induced actin polymerisation in vitro…" Katanaev VL, Wymann MP
http://jcs.biologists.org/content/111/11/1583.long
In my experience, GTP-gamma-S (from Sigma, G8634) works quite well. I used it both on purified proteins from bacteria and cellular lysates. In the second case, in order to maximize GTP loading of Rho-GTPases, I pre-treated the lysate with EDTA (5 mM) to get rid of endogenous loaded nucleotides. A second possibility would be to treat cells with a GTPase activator BEFORE harvesting and lysis: I used MnCl2 (1 mM) to activate integrins and I obtained a high level of RhoGTPases activation. After lysate extraction, I performed a GST-Rhotekin assay and the manganese effect on RhoA was really significant.
Hi Dmitri,
GTP-gamma is a good control but I wouldn't use GTP-gamma if I'm not 100% sure whether the activation of the myriad of GTPases that will get active when using GTP-gamma will not be affecting the signalling pathway I'm studying.
You should Check for some commertial Rho activation kits like the ones from cytoskeleton Rho activator 1 http://www.cytoskeleton.com/cn01 (it is basically Calpeptin) or Rho Activator 2 http://www.cytoskeleton.com/cn03 (which modifies the protein to turn it insensitive to GAP activity.
I have also used LPA (Lysophosphatidic acid) with good results.
Good luck!
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