Quantitating Westerns

rogier stugerNOstSPAM at cellbiology.uni-frankfurt.de.invalid
Mon Jun 26 07:37:03 EST 2000


Hiya,
you can expose a couple of films, scan 'em, and count the
blackness of the bands from non-saturated (scans of) films.
since the resolution of film is much better than an imager (there
are a zillion times more silver grains on a film than pixels in
an image) scanning films is more accurate than imaging. this only
holds if the film is not saturated, so make plenty of different
exposures.

keep in mind that western blotting efficiency is highest in the
center of the gel, and considerably lower in the outer lanes.
We always add a standard protein (or protein mix, like a cell
lysate) adjacent to every lane.
(like this: standard, two samples, standard,two samples,..., a
good way to run out of your postive control preps as fast as you
can), then divide the signal in every lane thru the signal of the
adjacent standard.


To count the signals, use imagequant if you have it. Or download
NIH image (Mac) or Scion image (PC version of NIH image) from the
web. Both programs are free.

Have fun,
Rogier Stuger
rogier AT bio.vu.nl

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