Dear Jacob, Nanodrop ultrafiltration sounds really fancy to me. I am afraid that I have no access to such equipment yet. Thanks for letting me know about this new technology.
Sincerely, Xuan Yang 2010/8/4 Jacob Keller <j-kell...@fsm.northwestern.edu> > 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 <pattisy...@gmail.com> 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 >> > >
