Sheep IgG's with Email

[Technical Assistance]

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
> beautifully.
> 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.

Mark Reichardt
Pierce Technical Assistance