western-quantitation??

Dr Engelbert Buxbaum eb15 at le.ac.uk
Fri Aug 28 15:04:09 EST 1998


Martin Offterdinger wrote:
> 
> Hi!
> I would like to know if you would consider it as science to quantitate
> bands obtained by enzymatic Western Blotting with a densitometer..

There are two problems here. One is that the response of a scanner to
the signal on the blot tends to be highly non-linear, the second is that
the bands have irregular shapes, making integration over the band area
tricky.

I have however had good success in immuno-quantitation of proteins by
using dot-blots instead (obviously you need to ascertain first that only
one protein in your sample reacts with the antibody). Samples were
affixed to a PVDF membrane (eg Immobilon P, Millipore) via a 96 well
filtration manifold (eg BioRad) by brief vacuum suction. I used to make
up the volume of each sample to 100 ul with PBS, to ensure that the
whole area of the dot was evenly covered by the antigen. The blott was
then blocked, developed with primary and secondary antibody (HRP
coupled) like a western blot and preincubated with chemoluminescent
reagent (eg Amersham). It was then placed in a special holder (taking
care to align the sample spots with the holes in the holder)  and
introduced into a 96 well chemoluminescent counter (eg Berthold).
Counting takes 1 s per well. 

I found this method very simple, sensitive and reliable, and the
log(signal) vs log(Antigen) plot was linear over 4-5 orders of
magnitude, but it is necessary to include a standard curve for each
blot.



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