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
