GHOST IMAGES!

[Mark Schweder]

In article <1994Mar5.221245.2377 at bay.cc.kcl.ac.uk>, udbs061 at bay.cc.kcl.ac.uk (MAHARAJAH) writes:
> Hello netters,
> I am having problems in staining gels using Nitroblue tetrazolium
> to study isozymes eg. alcohol dehydrogenase,fumarate hydratase etc.
> The major problem is I am supposed to get blue-violet bands at the regions
> of my isozymes instead i get the ghostimages of the the band and my
> gel get stained purplish blue. Has any body faced this problem and know the
> reasons.  I run native PAGE to study an array of isozymes.
> Any assistance will be well appreciated
> 
> Sri

I have a similar problem when staining for aldehyde dehydrogenase on native 
acrylamide gels and have found that incubating the gel with the appropriate 
reagents in the dark (black plastic bag) for about 30 minutes then rinsing 
the gel with water 3 or 4 times at 10 minute intervals and allowing the gel 
to incubate overnight in the dark will enhance visualization of the various 
isozymes. Unfortunately the entire gel does take on a dark blue appearance, 
however the location of the isozymes can be ascertained by the even darker 
blue bands. To photograph, place the gel on a white background (I use a 
piece of heavy white paper laminated with a plastic coating), illuminate 
from above (I use fluorescent light) and adjust f-stop and time for optimum 
contrast. I use an MP-4 Polaroid camera and type 55 film. Even when the 
difference between the background and bands can not be distinguished easily 
by eye, I can get very good contrast with the correct exposure.

All this does not help in reducing the blue background. I have tried 
lowering the NBT concentration by 1/2 and 1/2 again which didn't help. 
Any other suggestions or tips?
-- 
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   Mark Schweder                | There are three things that smell like fish!
   Medical Oncology             | One of them is fish...
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           904-392-9004         |         mschweder at gnv.ifas.ufl.edu
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                  DISCLAIMER: I know nothing about nothing.