Does anyone know what is the actual difference between CIP and EDTA method for exchanging the GppNHp (non-hydrolysable GTP) or GDP with RAS protein?
Dear Anamika,
CIP is Calf Intestinal alkaline Phosphatase, which specifically degrades GTP and GDP. Contrarily, EDTA chelates (in most cases) the Mg2+ ion bound to the nucleotide, thereby largely increasing its off rate.
See also this older question on RG with some hints for the EDTA method: https://www.researchgate.net/post/How_can_I_remove_the_GDP_and_GTP_from_GTPase_proteins
I hope this helps.
Best regards
Johann
Johann P Klare Thanks for your answer. And I read all the answer for this question https://www.researchgate.net/post/How_can_I_remove_the_GDP_and_GTP_from_GTPase_proteins as well. But still I am not able to understand If CIP degrades GTP and GDP then why we use CIP during exchange of the GTP/GDP with the purified RAS. Or you mean that it degrades free nucleotide in the solution during exchange.
Thanks
Anamika
Dear Anamika,
CIP unspecifally hydrolyses GTP and GDP...it just removes the terminal phosphate group from the nucleotide (or from DNA). Its mostly used to replace GDP (or GTP) with a non-hydrolysable GTP analogue like GppNHp or GTPgammaS. CIP (or CIAP) can also be used (with e.g. a subsequent gel filtration step) to obtain a nucleotide-free preparation of the G protein that can subsequently be "loaded" with the wanted nucleotide.
Best regards
Johann
Johann P Klare Hi I have a protocol according to which I need to incubate the purified RAS with GppNHp having 20U of alkaline phosphatase, 10uM of Zinc Chloride and 200mM ammonium sulfate for 2 hrs at room temperature. In this protocol they have mentioned that ammonium sulfate needs to be added in the last. I am quite confused, according to them do I need to add it after 2 hrs incubation or I have to add this at last to the mixture of (purified RAS+GppNHp+Zinc chloride and alkaline phosphatase) ? Please help me in this regard.
you have to add it as the last component to the reaction mixture - before the incubation time starts.
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