immunoprecipitation and 2D gels

[Alistair J Lax]

We are having problems visualising protein spots on 2D gels 
after immunoprecipitation, and would appreciate any 
suggestions. The protein is immunoprecipitated, and the 
beads then added to the first dimension buffer (urea and 
ampholites) and loaded onto the Biorad IEF cell. After the 
second dimension, the SDS PAGE gel is silver stained. This 
worked well for a couple of months, but all we get now are 
some lower molecular weight contaminants. Beads loaded 
straight onto SDS PAGE show the protein - either in blots 
or with silver stain. The early stages of the prep have 
protease inhibitors added, and we made all the solutions 
again after this problem appeared. We have tried adding SDS 
to the first dimension elution buffer without success. The 
protein is there when we elute the protein from the beads 
in SDS followed by removal of beads, that is it can be seen 
by SDS PAGE, but not in 2D gels.

Alistair Lax

alistair.lax at kcl.ac.uk
King's College London


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