Also depending on the detergent , If you have a detergent like decyl-maltopyranoside ( DM) or any other glycoside based detergent you can use the reaction with sulfuric acid and phenol followed by Absorption measurement to quantitate as detailed in
Anal Biochem. 2005 Jan 1;336(1):117-24. A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research. Urbani A, Warne T. Using a standard curve against the same detergent you can quantitate your detergent concentration in your final protein sample Hari On Fri, Oct 23, 2009 at 4:35 PM, Michael Matho <mma...@scripps.edu> 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 > To: CCP4BB@jiscmail.ac.uk > 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 > wei...@crystal.harvard.edu 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 > > pat.l...@drexelmed.edu
