densitometry on western blots

Jose de las Heras via (by josenet At
Thu Nov 23 15:56:54 EST 2006

<martinhoehne At> wrote in message 
news:1164299514.092168.323980 At
> Hello,
> I have some basic questions on densitometry and would be glad if anyone
> can point me to a good source to learn more or even answers some of the
> questions.
> 1) As I understand it the bands have to be non-saturated. Does this
> really mean that as soon as I have pixel values of 255 (on a 8-bit
> image) I can forget about doing a densitometric analysis?

on any digital image system, once you reach the limit of the dynamic range 
(256 for 8-bit, 65536 for 16-bit, etc) it doesn't make sense to use it.
You can get away with *a few* saturated pixels within the area you are 
measuring. Usually you would use the median (or mean) of the pixels in the 
area, or some other measurement, and having a few saturated pixels won't be 
too bad. But you should aim to avoid any saturation if you want accurate 

It's one little bug of mine, when you see figures with a loading control 
that's clearly saturated... and sure, they all look the same then! :-)


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