calculating protein conc. from extinction coefficients

Kyle Legate legatek at mcmail.cis.mcmaster.ca
Tue Sep 11 17:50:36 EST 2001


Hello all, I'd like your opinions on calculating protein concentration
from calculated extinction coefficients. I have read the Gill and von
Hippel paper (Anal. Biochem. 182:319-326 (1989)) and calculated the
extinction coefficient of a protein I work on at
www.basic.nwu.edu/biotools/ProteinCalc.html
and got 11170 M-1cm-1. Here's the trouble:
By calculating my protein concentration by the BCA assay I arrive at a
value of 0.83 mg/ml, but if I use the extinction coefficient method and
all of its assumptions, I calculate a concentration of 1.87 mM. For this
to be true I would need a BCA-calculated concentration of 52.36 mg/ml!
Here's the calculation (let me know if my units are off):

A280: .209 (I diluted the protein 1:100 in H2O, which gives me an adjusted
A280 of 20.9)

A280/E = [] in moles/litre

20.9/11170 = 0.001871 M = 1.871 mM

My concern is that extinction coefficients only look at Tyr, Trp and Cys;
what about His? My protein is in imidazole buffer, and I notice that a
dilute imidazole solution gives appreciable absorbance at 280 nm;
presumably histidine from a His tag will as well. Any comments?

... . . .  .  .  .    .    .    .     .     .     .      .      .      .
legatek at mcmaster.ca		Kyle Legate            legatek at hotmail.com

   Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
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