Well, you'll never loose the protein in the flowthrough if you don't discard the flowthrough.....which i never do until I'm sure about what happened to the protein. But yes, try different Mw cutoff concentrators until you find the highest one that works. Depending on the purification your sample may still contain lipids, which will make the micelle size even bigger compared to pure DDM. Also, some concentration of the detergent is not a problem. One tends to get more phase separation in crystallization trials with higher detergent concentrations but that does not necessarily prevent crystallization. If your protein is really stable and you're very worried about higher detergent concentrations, you can dialyze the protein against your buffer solution. Again try different cutoffs. You're not aiming for complete equilibration (which could take a long time), but to bring the concentration down via once or twice overnight dialysis. Personally we used to dialyze everything prior to freezing the protein but we have moved away from that w/o obvious effects on success rates.
Good luck, Bert ________________________________ From: CCP4 bulletin board [[email protected]] on behalf of Raji Edayathumangalam [[email protected]] Sent: Sunday, July 14, 2013 1:47 AM To: [email protected] Subject: [ccp4bb] Concentrating purified membrane protein Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
