"Halos" in Western Blots; What are they?

John S Walker jsw9c at virginia.edu
Mon Mar 9 16:57:01 EST 1998


G'day,

I'm a newbie to doing Western blots. At present I'm trying to optimise a
technique for quantifying the percentage phosphorylation of a protein.
The protocol i'm following consists of an isoelectric focusing gel that
is then transferred to a nitrocellulose membrane.  The membrane is then
exposed to primary and secondary antibodies.  The detection system uses
enhanced chemiluminescence with a horseradish peroxidase linked
secondary.  The light is detected using x-ray film.  At present we are
having problems with linearity of response and a steep response curve
(i.e. we only go from detection limit to saturation over a five-fold
range of protein concentrations) In addition, and this brings me to my
question, we have halo's around the detection bands, i.e. a light band
just above the more heavily stained 'normal' location of the band.  I've
read some accounts that say that this is due to protein overloading and
some that say it's due to excessive antibody.  Can anybody give me a
clear answer as to what causes these halo's and why?  I don't think that
this is a 'real' band.


John Walker
Dept of Physiology
University of Virginia



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