I have to perform an NMR experiment on a sample to study the hypothetical formation of a protein-DNA complex.
The switch from a high salt concentration buffer is needed (in which there is the binding reaction) to a low concentration buffer in order to acquire the NMR spectrum.
I'm wondering if the centricon centrifugation step required in order to exchange the high concentration to the low concentration buffer could damage the hypothetical DNA-Protein complex?
Personally I do not like using Centricons for buffer exchange. I am always afraid that repeated concentration (and sometimes one just forgets the sample and it becomes quite concentrated) leads to precipitation. I sometimes also lost some protein presumably because it adsorbed to the membrane (try different types of membrane). ALternative ways of changing buffer are dialysis (use of dialysis buttons enables you to dialyse quite small sample volumes - you can make your own dialysis button by cutting an Eppendorf in the upper part, open it, put your sample in the inside of the lid, place a dialysis membrane on top and fix it with the remaining part of the tube) or use of small desalting columns (NAP5, NAP10 for 0.5 or 1 ml sample volume). Your sample will be slightly diluted but a single concentration step on a Centricon will allow you to obtain the desired volume.
Hope that helps
Beate
Dialysis is the way to go and minimises the potentially large losses of the complex in the pores of the Centricon membranes
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