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Regarding concentrating detergents w/ MWCO concentrators - may I suggest the following reference: Refractive index-based determination of detergent concentration and its application to the study of membrane proteins Pavel Strop and Axel T. Brunger Protein Sci. 2005 August; 14(8): 2207–2211. Michael Matho wrote: > Weikai, > > We did it using NMR but you asked for a simple way so I guess I'm out > of topic. > > Anyway, since I believe it is the most accurate method, here it is: > using a high detergent concentration stock solution you can assign > resonance peaks to your detergent molecule bonds. > > Then you can set up a standard curve using different known detergent > concentrations (for example from 10% down to 0.1%) by calculating the > surface of your peak(s) which is directly related to your detergent > concentration. > > Each time you need to know the concentration of a new sample, you just > need to record the peaks, and use the three-click rule to deduct the > unknown value. > > As a colleague answered you earlier, we noticed that a 50kDa cutoff > withheld a lot of detergent during concentration process and > consequently your final concentration might increase significantly. > For example we started with 0.25% DES and noticed increases of above > 1%. Of course this will depend on the concentration factor. > > This did not happen when using a 100kDa cutoff, and DES concentration > remain pretty much constant. > > Now, it will depend on your system: what detergent you are > using, since micel size and CMC are obviously the critical parameters > here -- but also what maximal cutoff you can use w/o loosing your > membrane protein in the flow through... > > Good luck, > Michael > > ----- Original Message ----- > *From:* Patrick Loll <mailto:[log in to unmask]> > *To:* [log in to unmask] <mailto:[log in to unmask]> > *Sent:* Friday, October 23, 2009 1:12 PM > *Subject:* Re: [ccp4bb] measure detergent concentration > > I'll second this. We've done this as an exercise in NSLS Membrane > Protein Crystallization workshop for a few years, and it works > like a charm. You can stain in a warm iodine chamber and visualize > by scanning the TLC plate on a garden variety scanner (we use an > inexpensive Canon LIDE that probably cost less than USD 60 five > years ago). We quantify the spot intensity with NIH Image or > equivalent, and get lovely linearity down to the CMC, spotting > only 1 uL of sample--so we haven't seen any need to concentrate. > > On 23 Oct 2009, at 3:41 PM, Edward A. Berry wrote: > >> Only easy if you happen to have silica gel TLC plates and >> a chromatography jar lying around, perhaps from some >> phospholipid analysis: >> >> A strategy for identification and quantification of >> detergents frequently used in the purification of membrane proteins >> Laura R. Eriks, June A. Mayor, and Ronald S. Kaplan >> Analytical Biochemistry 323 (2003) 234–241 >> >> This paper recommends spotting on a TLC plate and running >> beside standard amounts of the same detergent. From intensity/size >> of the detergent spot after developing you can bracket the detergent >> concentration. (And by the way they found that detergents are >> concentrated by ultrafiltration). To increase sensitivity, >> speedvac a volume too large to >> spot on the plate, dissolve the residue in Me0H. >> >> Ed >> [log in to unmask] <mailto:[log in to unmask]> wrote: >>> Hi Folks: >>> After concentrating a membrane protein, is there a (easy) way of >>> measuring >>> the detergent concentration in the sample? >>> Regards, >>> Weikai > > --------------------------------------------------------------------------------------- > > Patrick J. Loll, Ph. D. > > Professor of Biochemistry & Molecular Biology > > Director, Biochemistry Graduate Program > > Drexel University College of Medicine > > Room 10-102 New College Building > > 245 N. 15th St., Mailstop 497 > > Philadelphia, PA 19102-1192 USA > > > (215) 762-7706 > > [log in to unmask] <mailto:[log in to unmask]> > >