calculating protein conc. from extinction coefficients
jphilo_nospam_ at mailway.com
Wed Sep 12 16:23:51 EST 2001
Your calculation of the molar concentration is correct. To know how that
translates to mg/ml one needs to know the molecular weight, which you didn't
mention, but your 52 mg/ml number will be correct for a 28 kDa protein.
For most proteins the absorbance at 280 for 1 mg/ml and 1 cm path is
0.5-1.5, so your A280 of ~20 would indeed typically indicate a concentration
of tens of mgs/ml.
With regard to the accuracy of these calculated extinction coefficients,
your concern about not considering His in the calculations is not
valid---His has no significant absorbtion at 280 nm. In my experience, and
that of many others, these calculated extinction coefficients are usually
accurate within 5 percent (and often better) even for native, folded
proteins (and therefore far more accurate than any dye binding assay). The
calculated values are certainly are never off by orders of magnitude!
Having said that, I think your problem indeed has to do with background
absorbance of your buffer containing imidazole. You need to take a reading
on an aliquot of your buffer alone and subtract its A280 from that of your
protein solution. You have aggravated the buffer absorption issue
tremendously by your high dilution of the protein solution. If your BCA
assay is correct, the A280 of your undiluted sample should be only ~0.33.
You should try measuring that directly.
'Hope this helps.
Alliance Protein Laboratories
"Kyle Legate" <legatek at mcmail.cis.mcmaster.ca> wrote in message
news:Pine.SOL.4.33.0109111810520.24304-100000 at mcmail.cis.mcmaster.ca...
> 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
> 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
> moon musick:ritual:IDM:experimental(electronica):minimalism:glitch
> . . . . . . . . . . . . . . . ...
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