Dear Xuan,
I am not certain, but I think that Jacob was referring to a spectrophotometer called a Nanodrop. It is available from ThermoFisher Scientific and can provide absorbance data on as little as 2uL of sample. I think that if you have access to a Nanodrop, and use Ultrafree 0.5mL concentrators, you can achieve the results that Jacob described. Hope that helps, Bryan From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Xuan Yang Sent: Wednesday, August 04, 2010 9:46 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] I-TASSER predicts NADPH binding, need to confirm with experiment 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 -------------------------------------------------------------------------- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
