I like nanodrop ultrafiltration: concentrate your protein to the highest stable concentration possible
figure out what is the lowest possible robustly-detectable nadph signal on your nanodrop combine the two in such a way in the top of a microcon of appropriate MWCO to acheive the highest possible protein concentration with the lowest possible nadph concentration. Take a baseline spec reading before spinning. spin long enough to get enough flowthrough to measure on the nano (~10uL is plenty.) Flowthrough should be the free nadph concentration L. Total L should be known, as well as total P, so you can figure out bound concentrations PL easily. you should probable do this in triplicate or so, with appropriate controls. I found 50uL/microcon to be a good balance of pipette-ability and economy of protein. If you want to get fancier, you can do more samples varying concentrations (or do some other more sophisticated method.) Jacob On Wed, Aug 4, 2010 at 3:10 AM, Xuan Yang <[email protected]> wrote: > Dear All, > > 3D structure modeling server I-TASSER predicts a binding site for NADPH and > I want to test this prediction. What would be the nice quick way to tell > whether this protein bind NADPH or not, when I have a lot of recombinant > protein? > > Sincerely, > > Xuan Yang >
