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confirming nucleotide state of isolated G protein


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#1 mmt

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Posted 05 February 2009 - 06:57 PM

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.

#2 rkay447

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Posted 06 February 2009 - 06:52 AM

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.




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