confirming nucleotide state of isolated G protein - (Feb/05/2009 )
hi, I am looking for a way to tell the state of the G protein I am working with. you know G proteins are found in GTP or GDP bound states. I need a way to tell between these two. I am loading my protein with GTP the way they do in the literature. but I want to have an idea of what percent of my protein is actually in the state I want. does anybody have an idea? thanks in advance.
Use radiolabelled GTP. You can calculate from specific activity the mols of protein that are GTP bound and then you can also calculate the hydrolysis by loss of counts. I also saw that perkin elmer has a system for fluorescent detection rather than radioactive. I'm sure if you do a literature search you will find groups who have done exactly what you are hoping to accomplish. You can then look at the protocols they publish or reference.