polyclonal antibody purification

Brad Nicholson Brad_Nicholson at hlthsci.med.utah.edu
Fri Jan 31 07:09:04 EST 1997


In article <5cgh64$1lb at news.acns.nwu.edu>, ageorge at casbah.acns.nwu.edu wrote:

> I have been having problems trying to purify a polyclonal antibody raised in 
> rabbits. The antigen was an e.coli expressed protein. There is a lot of
background.
> does anyone out there know what is the best method.
> Thanks
> -- 
> ANNE GEORGE
> Northwestern University, Evanston, IL.   USA
> ageorge at casbah.acns.nwu.edu

Hello Anne,

Things that I have used in order of effectiveness.

I'm guessing that you are getting some non-specific E. coli reactive
antibodies that are making your blots look bad?  If that is the case, you
can make a column of E. coli that aren't expressing your protein of
interest, conjugated to a solid support (Reacta-Gel from Pierce?).  You
use that to pull out the antibodies that are making your blots look bad.  

Alternatively, you can make an acetone powder from your strain of interest
and use that to subtract the non-specific antibodies.  This is cheaper and
a little faster, but it didn't work as well in my hands.  YMMV.

If you are screening a phage expression library and getting unacceptable
background you could also set up a bunch of filters that do not have your
clone of interest (vector phage alone or a known clone) use these to pan
out the non-specific antibodies.

Good luck,
Brad



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