Hold on--did you add free NADPH into the top of the concentrator, and spec the sample pre-spinning? You need to be sure that you can see the NADPH in the sample before you spin it, because you are looking for a *loss* of NADPH absorbance in the flow-through.
A similar method uses a very low level of NADPH in the running buffer equilibrated on an SEC column. When you run your concentrated protein sample through, you should see a negative peak somewhere corresponding to the NADPH "sucked up" by your protein, and a robust NADPH peak co-eluting with your protein. Jacob ----- Original Message ----- From: Xuan Yang To: [email protected] Sent: Tuesday, August 17, 2010 4:17 AM Subject: [ccp4bb] Summary: I-TASSER predicts NADPH binding, need to confirm with experiment Dear all, Here comes a late summary for the issue about NADPH. Considering the specific absoption at 340nm, UV spectrum was suggested as a nice way to check NADPH. Thanks to Vellieux Frederic. I recored the UV spectrum of freezed protein and fresh sample from 400nm to 240nm. The results were negative. No 340nm peak was observed at all (attachment 1 series). Jacob Keller & Markus Seeliger suggested a different way employing ultrafiltration. If the protein binds NADPH, the flowthrough should contain a significant amount of free NADPH for 340nm detection. I did not get it at first, now I consider it as a smart way. Unfortunately, the results were also negative (attachment 2). Andrzej Lyskowski and Jürgen Bosch suggested another powerful way called DSF/Thermoflour. The basic idea is that a relevant cofactor should change the Tm of protein during heat denaturation. I found Sypro Orange and test the Tm shift of my protein in NADPH via RT-PCR. The results were also negative (attachment 3). But it should be interesting to test whether other cofactors might be effective. Now I conclude that the prediction was wrong and the protein does not bind NADPH. Thanks for everybody involved. ***************************Jacob's precedure*********************** Jacob's precedure: "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.)" *******************DSF/Thermoflour Protocol********************* DSF/Thermoflour Protocol: http://www.nature.com/nprot/journal/v2/n9/abs/nprot.2007.321.html. 2010/8/4 Xuan Yang <[email protected]> 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 ******************************************* Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [email protected] *******************************************
