Re: [ccp4bb] Concentrating purified membrane protein

[El Arnaout, Toufic]

Hi, That is true, Michael.. can't remember the reference, but in some study they found that even if you use a higher MWCO (for example 100 kDa) with DDM (micelle 65-70 kDa), there is still 70-80 % retained detergent in the protein sample. Raji, another thing I would like to say is that the right MW cut-off may depend on the protein, the detergent micelle, but also the bound detergent molecules per protein. It could be 40-50 molecules of DM let's say for protein A, but 80-90 molecules for protein B. You can quickly evaluate the concentration step for your protein using SDS-PAGE or UV abs, and for the detergent there are many methods like colorimetric assays, TLC.. Best wishes t toufic el arnaout School of Medicine - 660 S Euclid Ave Washington University in St. Louis St Louis, MO 63110, USA From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of R. M. Garavito [rmgarav...@gmail.com] Sent: Sunday, July 14, 2013 11:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Concentrating purified membrane protein 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:  rmgarav...@gmail.com **************************************************************** 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 <r...@brandeis.edu> 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