Question about quantitating western blots.
Christopher W Kafer
ckafer at iastate.edu
Fri Dec 7 14:08:38 EST 2001
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!
"Emir" <REMOVEem at unforgettable.com> wrote in
news:kS5Q7.235$t4.11554 at news.uchicago.edu:
> As others mentioned here, the most important is running several (min
> 3-4) lanes with different concentrations of your protein to determine
> linear range of detection by making standard curves of sum of pixel
> values in a spot / quantity of protein. One hint on digital
> quantitation: adjust your camera or scanner so that background
> intensity (do the black and white scan) is equal or near 0 pixels.
> There is only 256 max possible pixels of intensity, and this is the
> major limitation of any digital analysis, because that makes linear
> range of detection very narrow.
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