Raji, One point the most people forget about is that whenever you concentrate any detergent-solubilized membrane protein is that you will ALWAYS concentrate the detergent. So regardless of the MWCO, if the protein-detergent complex concentrates, the overall detergent concentration also increases. What you want to shoot for is balancing protein loss with obtaining a sample having minimal EXCESS free detergent. While a concentration step with a Ni-column followed by dialysis will work, so will a wise choice of concentrator. One trick to moderate high excess detergent is just to avoid the need for a many fold concentration where you will really concentrate the free detergent. Nonetheless, we have used concentrators effectively, and a Ni-column followed by dialysis as well, but if you still have too much free detergent, you can always use a spin desalting column with G-25 sephedex to bring remove detergent down to a nominal concentration. In all cases, you will lose some protein.
Good luck, Michael **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected] **************************************************************** On Jul 14, 2013, at 10:29 AM, Raji Edayathumangalam wrote: > Thanks everyone for your responses. I definitely plan to save the flowthrough > so we'll see what happens. My protein has a His tag and I did consider doing > an affinity step for concentration except I do not want to have imidazole for > some functional assays that I need to carry out with the protein. Just > occurred to me that I could simply dialyze out the imidazole after the > affinity step. > > Thanks again! > Raji > > > On Sat, Jul 13, 2013 at 8:47 PM, Raji Edayathumangalam <[email protected]> > wrote: > 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 > > > > > -- > Raji Edayathumangalam > Instructor in Neurology, Harvard Medical School > Research Associate, Brigham and Women's Hospital > Visiting Research Scholar, Brandeis University >
