Question about quantitating western blots.
Ian A. York
iayork at panix.com
Fri Dec 7 15:44:05 EST 2001
In article <Ws8Q7.7697$va.3358906 at news2.rdc1.mi.home.com>,
Christopher W Kafer <ckafer at iastate.edu> wrote:
>Thanks for the input, it's greatly appreciated! One more question, if
>what we really want to know is the relative amount of protein between
>samples, is a standard curve on the gel necessary? We arent interested in
>absolute amounts of protein in each sample. ie if we scan the blot and
>quantitate the bands, can we say there is 2 fold more protein on day 2
>than on day 1 for example?
You absolutely can not do this. (Well, you can *do* it, but the results
will be meaningless.) The intensity of a band is not propertional to the
amount of protein in the band, on a western. There are too many
non-linear processes involved.
What you can do, to get semi-quantitative data, is this: Make serial
dilutions of your samples, say 2-fold or three-fold. Run then all on the
same gel. Quantify band intensity. Now, bands of the same intensity
should have roughly the same amount of protein.
But you'll also see that you don't get neat two- or three-fold steps in
band intensity (or if you do, it's over such a small, and variable, range
that it's no general use).
Ian York (iayork at panix.com) <http://www.panix.com/~iayork/>
"-but as he was a York, I am rather inclined to suppose him a
very respectable Man." -Jane Austen, The History of England
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