The easiest way to measure detergent concentration is to make of use of surface tension properties - simple, quick and effective. The method is described in the following paper:
http://www.ncbi.nlm.nih.gov:80/pmc/articles/PMC1367029/ Good luck. Chris. 2009/10/23 Ezra Peisach <[email protected]>: > 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:[email protected]> >> *To:* [email protected] <mailto:[email protected]> >> *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 >>> [email protected] <mailto:[email protected]> 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 >> >> [email protected] <mailto:[email protected]> >> >> > -- Dr. Christopher J. Law, Lecturer in Biochemistry, Queen's University Belfast School of Biological Sciences Room 185, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL. Tel: 028 9097 2071 (Direct line) Mob: 07913 078410 Web page: http://www.qub.ac.uk/schools/SchoolofBiologicalSciences/Staff/DrCJLaw/
