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 >
