Here is also a very effective method: 1 Gill, S. & Hippel, P. v. Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry 182, 319-326, (1989).
On Thu, Jun 16, 2011 at 5:56 PM, Filip Van Petegem < filip.vanpete...@gmail.com> wrote: > 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/ > -- Scott D. Pegan, Ph.D. Assistant Professor Chemistry & Biochemistry University of Denver Office: 303 871 2533 Fax: 303 871 2254
