A convenient fast way is the earlier mentioned Edelhoch method, as described in this paper which is referenced on the popular Protparam tool:
http://onlinelibrary.wiley.com/doi/10.1002/pro.5560041120/pdf Filip On Thu, Jun 16, 2011 at 4:45 PM, aaleshin <aales...@burnham.org> wrote: > Mischa, > You intrigued me. What is the experimental technique for the Extinction > Coefficient measurement (which requires knowledge of protein > concentration)? Let me guess, Bradford? Protein evaporation and weighing? > > Alex > > > On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote: > > With respect to the Edelhoch method and the ProtParam server, I would > strongly recommend determining extinction coefficients experimentally and > not rely on the ProtParam values. The reason is that the underlying > extinction coefficients in the formula used by ProtParam and referenced > there are statistical averages. They may or may not be valid for a given > protein. I have seen differences of more than 20% between the "theoretical" > and "experimental" extinction coefficients, particularly for proteins with > few Trp and Tyr residues. When relying on relative concentrations, this > inaccuracy is not detrimental, but when absolute concentrations are needed > (CD, AUC, ITC, any binding experiment, etc.), such a difference would be > considered huge. Determining an extinction coefficient experimentally takes > but a few minutes. > > Cheers! > MM > > > On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote: > > Totally support the statements below. We have had several proteins with > A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in > the Nanodrop or whatnot to measure the concentration. > > > Before purchasing the Nanodrop we used a Hellma TrayCell and a "normal" > UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell is > 2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but > Nanodrop is a lot more convenient to use for high concentration quick > measurements (especially if you need to measure several things in > succession), so you get what you pay for. > > > Petr > > > P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). > That plus the Nanodrop are two essential and synergetic tools of a protein > chemist/crystallographer. > > > On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote: > > > > Bradford is an assay, Nanodrop is a spectrophotometer. > > Both the A280 and Bradford methods are strongly dependent on > > amino acid composition, so unless you correct A280 for that > > as mentioned by Filip, either one is semiquantitative. > > Occasionally you come across a protein with no tryptophan > > which will have a much lower extinction coefficient. > > Try making a 1 g/l solution of gelatin (collagen?) > > and see what its A280 is! I noticed recently the > > "protparam" tool at http://ca.expasy.org/cgi-bin/protparam > > estimates the extinction coefficient given a sequence. > > > > > David Briggs wrote: > > ~~~ > > > I wouldn't touch Bradford with a barge-pole. I've found it to be > > wildly inaccurate for certain proteins I've handled, where as the > > OD280 measurements have been fine. > > > One wonders what does "fine" mean, like same as with Biuret or > > Kjeldahl nitrogen, or solution made up by weight? > > > ----------------------------------------------------------------------- > Mischa Machius, PhD > Director, Center for Structural Biology > Assoc. Professor, Dept. of Pharmacology > Member, Lineberger Comprehensive Cancer Center > University of North Carolina > 4079 Genetic Medicine > CB#7365 > 120 Mason Farm Road > Chapel Hill, NC 27599-7365, U.S.A. > tel: +1-919-843-4485 > fax: +1-919-966-5640 > email: mach...@unc.edu <mach...@med.unc.edu> > > > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
