co-immunoprecipitation problem

Bart Sefton Bart_Sefton at QM.Salk.Edu
Fri May 26 13:45:08 EST 1995


In article <3q0ugr$rdc at sifon.cc.mcgill.ca>, bg2z at musicb.mcgill.ca wrote:

> I am using a rabbit antibody to immunoprecipitate a protein under 
> co-immunoprecipitation conditions, which should, and does bring down a
complex 
> containing another protein (HA tagged) against which I have a mouse Ab 
> (12CA5). The problem I have is that my initial immunoprecipitating rabbit Ab 
> is recognized on the nitrocellulose by the secondary Ab I use to detect 12CA5 
> (Goat anti mouse HRP- I detect using ECL) and the heavy chain migrates almost 
> exactly where the tagged protein is located. I know my protein is there 
> because there is always extra signal right above the IgG heavy chain, but it 
> would look much nicer if there was a way to degrade, or get rid of 
> specifically the IgG heavy chain. Other than running really loose gels
(7% and 
> less) is there a way to get rid of that fat whopping IgG band? any
advice will 
> be greatly appreciated
> 
> Dino De Angelis, McGill Biochemistry

Iodinated protein A will sometimes, but only sometimes, distinguish
between your detecting antibody and a precipitating antibody (which has
been totally denatured).  Another approach is to run the samples without
reduction.  Under these conditions, the precipitating antibody runs as a
150 K species.  The best way to do this is to include 50 mmolar
iodoacetamide in your lysis and washing buffers and then use a sample
buffer that lacks both mercaptoethanol and DTT and contains instead 50 mm
iodoacetamide.  It is very important that the iodoacetamide be fresh and
high grade.  Additionally, due to the very high concentration of
mercaptoethanol in most sample buffers and its rapid rate of diffusion, it
is necessary to flank a non-reduced sample with other non-reduced samples
or lanes loaded with only non-reducing sample buffer if you are also
analyzing reduced samples on the same gel.  This way, the iodoacetamide in
the adjoining samples consumes any diffusing mercaptoethanol.



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