Hi All, Does anyone know what is the actual difference between CIP and EDTA method for exchanging the GppNHp (non-hydrolysable GTP) or GDP with the purified RAS protein?
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 hr 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)? Since this protein and its mechanism is new to me so not sure what to do. After reading some papers also I am not able to understand which method is good for exchange and how these reactions behave? Please help me in this regard. -- Anamika Singh Post-Doctoral Fellow Silberman Institute of Life Sciences Hebrew University of Jerusalem, Israel No: 054-294-8036 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
