I see, by the experimental determination of the extinction coefficient you mean correction for the difference between unfolded (which can be computed accurately) and folded proteins. Am I right?
Sorry for making this topic viral... Alex On Jun 16, 2011, at 5:06 PM, Machius, Mischa Christian wrote: > The method is that by Edelhoch, mentioned a couple of times already in this > discussion. It's also described in the paper by Pace et al., the same paper > that the formula in ProtParam is from (ProtParam does not use the values > determined by Gill & von Hippel). Last time I looked into this, the consensus > was that the Edelhoch method is the most accurate method for protein > concentration determination; more accurate than dry-weighing plus N-terminal > sequencing, etc. > > MM > > > On Jun 16, 2011, at 7:51 PM, aaleshin wrote: > >> 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 >>>> >>> >> > > ----------------------------------------------------------------------- > 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 >
