Sorry for misprint, I meant evaporating water from a protein solution... On Jun 16, 2011, at 4:45 PM, aaleshin 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 >> >
