Re: [ccp4bb] Protein concentration form chromatograms
If using Unicorn, 1)Open your chromatogram in Evaluation window. 2)Go to Operations-> Pool 3)Choose which baseline estimation suits you, define the pools (numbered rulers that appear under the chromatogram) 4)type in the path length (2 or 10 mm for UV-900 and 2 mm for UPC-900) 5)type in the mass extinction coefficient (Molar extinction coefficient/Mw in daltons) 6) get your answer from the table at the bottom of the screen The reading will make sense for pure protein. Also note that for the most accurate result you need to know the exact path length which varies a little bit from cell to cell (or so they say). Regards, Dmitry On 2014-01-15, at 10:09 AM, Karel Chaz wrote: > Dear all, > > A question for the biochemistry-inclined folks in the bb; how do I calculate > protein concentration of chromatography fractions, starting from Abs280 from > the UV monitor? I know I could figure it out myself if I really tried, but > why bother when I have access to so many brilliant minds > > > Thanks to all, > > K smime.p7s Description: S/MIME cryptographic signature
Re: [ccp4bb] Protein concentration form chromatograms
I don't think it possible for protein purification purpose. There would be no linear correlation between A280 and the protein concentration. For pure protein analysis, as for minor amount protein used, it is possible. Acoot From: Karel Chaz To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 15 January 2014 11:09 PM Subject: [ccp4bb] Protein concentration form chromatograms Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K
Re: [ccp4bb] Protein concentration form chromatograms
Thanks for all the replies. And I would not call this laziness, rather crowdsourcing I should have added a few more details; it is easy to export from say Unicorn to excel, a list of pairs of values, Abs280 vs elution times, with which one can recreate the chromatogram. I wanted to use these for my calculations. K On Wed, Jan 15, 2014 at 10:49 AM, Evgeny Osipov wrote: > I am not sure, but probably path length not 1 cm in a detector cuvette . > You must refer to your HPLS system manual for this value > 15.01.2014 19:09, Karel Chaz пишет: > > Dear all, >> >> A question for the biochemistry-inclined folks in the bb; how do I >> calculate protein concentration of chromatography fractions, starting from >> Abs280 from the UV monitor? I know I could figure it out myself if I really >> tried, but why bother when I have access to so many brilliant minds >> >> >> Thanks to all, >> >> K >> > > > -- > Eugene Osipov > Junior Research Scientist > Laboratory of Enzyme Engineering > A.N. Bach Institute of Biochemistry > Russian Academy of Sciences > Leninsky pr. 33, 119071 Moscow, Russia > e-mail: e.m.osi...@gmail.com >
Re: [ccp4bb] Protein concentration form chromatograms
I am not sure, but probably path length not 1 cm in a detector cuvette . You must refer to your HPLS system manual for this value 15.01.2014 19:09, Karel Chaz пишет: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] Protein concentration form chromatograms
On 1/15/14, 9:34 AM, Matthew Franklin wrote: - I usually approximate the peak as a triangle, so that the total peak area is 1/2 height (absorbance maximum) x base (the number of ml in your pool) GE's UNICORN for AKTAs (and probably the new Biorad FPLC software) integrates and tells you the area under whatever peak. Baselines and peak boundaries are user adjustable.
Re: [ccp4bb] Protein concentration form chromatograms
Hi Karel - To add to what Jurgen said, a few points on the measurement of the protein peak from the chromatogram. - I usually approximate the peak as a triangle, so that the total peak area is 1/2 height (absorbance maximum) x base (the number of ml in your pool) - If your peak is a strong one, watch out for non-linearity in the absorbance measurement - my Akta UV monitor doesn't give a reliable reading once the A280 goes above 1.8. This will cause you to underestimate your total protein amount. - You also need to apply a correction factor since your UV cell path length isn't 1 cm. You could look up what the path length is in the manual, but the easiest way to do this is to compare UV readings for a set of fractions from your FPLC monitor and a standard spectrophotometer. Figure out the ratio of the two (it'll be a simple whole number, probably 2 or 5, or maybe 1 if your UV monitor does the correction automatically), then put it on a post-it next to the UV monitor so you won't have to do this again. Now multiply your chromatogram's integrated peak area by this factor to give you the "standard" (1 cm path length) A280 measurement. Hope that helps, Matt On 1/15/14 10:09 AM, Karel Chaz wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Protein concentration form chromatograms
Step1: visit Protparam tool and C&P your sequence, scroll down until you find the extinction coefficient part. If OD280 is not close to 1 then make sure to take that into account in your chromatogram say it is 0.7 step2: look at your mAU's If your peak is 1000 mAU then you have 0.7 mg/ml in that fraction Step3: you are pretty lazy google for it Jürgen On Jan 15, 2014, at 10:09 AM, Karel Chaz mailto:karel.c...@gmail.com>> wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
[ccp4bb] Protein concentration form chromatograms
Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K
