Question about quantitating western blots.
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Fri Dec 7 14:31:16 EST 2001
In my experience, the signals can be vary non-linear, and dependent on a
number of factors, including the nature of the Ab, so a 2-fold difference
in signal might not mean a 2 fold difference in protein. If you don't want
to run a standard curve, I think you may at least have to run various
amounts of the samples you wish to compare. Then you'd be able to say
something like 5 ug of total protein on day 2 had the same signal as 10 ug
of total protein on day 1, so day 2 had 2-fold more of your protein of
interest. However, if you just loaded 10 ug of each sample and saw signals
that were 2-fold different, I don't think you could draw the conclusion
that there was 2-fold more protein. Does that make sense?
Hope this helps.
>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?
>Or is the standard curve necessary to be certain that the measured pixel
>intensity of the samples falls within the linear range of the scan?
>Thanks again for the help!
Michael L. Sullivan, Ph.D
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706
(608) 264-5144 Phone
(608) 264-5147 Fax
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