Western with sheep-IgG's

room 103 at bham.ac.uk
Mon Jun 24 10:41:41 EST 1996

Hi folks,
I'm having big problems using antibodies raised in sheep. Antibodies were
raised against a 6xHistidine tagged fusion protein of a human cell cycle
protein.Fusion protein was produced in E.coli strain M15 and purified
under denaturing conditions.
The problem is when I develop Western blots of whole human cell extracts
using the ECL system the background is so high even at dilutions of 5000
that any specific signal is lost.However this is not a problem when
testing the antibodies on Westerns against the fusion protein ,which work
The blocking system I'm using is skimmed milk (5%) and Tween 20 (0.1%).
All wash buffers contain PBS and Tween.
Is this system okay for sheep IgG's?
Can I get rid of this background with a different blocking system?

Is there any advice on using these sheep antibodies which doesn't involve
pouring them down the sink.

                  A desperate PhD student....Cheers

                           Andy Sutcliffe

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