quantitative western blots

Eric P. Ingersoll ei00+ at andrew.cmu.edu
Mon Nov 4 18:20:26 EST 1991


David Rintoul asked for references on performing quantiative western
blots.  I know of several methods that might be useful.  You could of
course use an iodinated secondary antibody or 125I-protein A to probe
your blots and expose to X ray film.  The autorad could then be scanned
on a standard densitometer.  There are two nonradioactive methods that I
am familiar with.  The first is to take a photograph of your
phosphatase/peroxidase on large format direct positive film.  The
"negative" (which is really a positive image) can then be scanned on a
densitometer.  A preferable nonradioactive method might be to use the
Lumi-Phos kit sold by Boehringer Mannheim.  This is a chemiluminescent
substrate for alkaline phosphatase.  All you do is wet the membrane with
the substrate and expose to X ray film.  In a few minutes you develop
the film and have an "autorad" of your data that can be scanned.  It is
a very sensitive assay (more sensitive than the NBT/BCIP substrate) and
it is very rapid.  The Lumi-Phos is a rather expensive reagent though
($260 for 100 ml).  I hope some of these suggestions are helpful to you.
 Give the Lumi-Phos a try and good luck!

Eric Ingersoll
Internet:ei00+ at andrew.cmu.edu



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