Sheep IgG's with Email
TechAssist at pierce.geis.com
Tue Jun 25 15:03:17 EST 1996
room 103 wrote:
> 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 A.sutcliffe at bham.ac.uk
Did you purify your goat IgG? Background problems can often be eliminated by
purifying polyclonal IgG over a Immobilized Protein A column.
Also, if you are using an avidin-biotin system for detection, milk should be
avoided as a blocker since it can contain free biotin. SuperBlock would be a
better blocker in this case.
It is also important to use a high quality secondary antibody-HRP conjugate at
the correct dilution to avoid background problems.
Pierce Technical Assistance
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