Hi Raji, DDM has quite a large micelle so you might be alright with a 100kDa cut off concentrator, I've had the same experience with a 30kDa protein in LDAO being comfortably contained by a 100kDa concentrator. I would try with a small amount of you sample and see if significant amounts of protein are found in the flow through. Is your protein tagged? if so you could bind it to a small volume (1ml) affinity column, exchange it into the lowest DDM conc. it is stable in (0.01% ?) and elute with a very steep gradient. If this didn't give you the desired concentration at least it would minimize the volume to be concentrated (and thus the final detergent concentration). You could also try and intermediate cut off size concentrators you have available (20,30,50kDa etc) until you find biggest one that retain the sample.
Good luck Rhys Grinter ________________________________________ From: CCP4 bulletin board [[email protected]] On Behalf Of Raji Edayathumangalam [[email protected]] Sent: 14 July 2013 01:47 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
