Cleaning up primary antibodies??

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Thu Dec 14 10:28:13 EST 2000


Neal Robert Melvin <nrmelvin at ucalgary.ca> wrote:
:Is it possible to 'clean up' a primary antisera by incubating with protein
:A, and then somehow gently eluting the IgGs from the protein A to be able
:to use?? What is the best way to go about this??
:

Yes, it is possible of course. The question is whether you will 
benefit from doing it at all. In some applications purified IgG vs
crude serum will make a difference, in some won't. 
If, for example, you are going to use them for Westerns, then
Protein A purification won't make a difference (you will purify 
all IgGs - those against your antigen and all the other IgG that 
contribute to "non-specific" signal). 

The only way to really clean up polyclonals with regard to 
a particular antigen binding is to do affinity chromatography
on immobilized antigen. Doing Protein A column before 
antigen column (good way to keep your it cleaner ans last
longer) or after (great way of concentrating IgG) is a good
option. Here is what I routinely do to get ~99% pure affinity 
purified IgG for antigen neutralization studies: 

Serum -> antigen column -> elution with 3.5M MgCl2 pHed
to 6.5 (= Pierce's "gentle elution buffer"; this is the way to
go if the antigen is a protein that does not like low pH) -> 
dialysis -> Protein A column -> elution with 100 mM glycine 
pH 2.5 into 1/10V 1M Tris, pH 8.0. Works like a charm. 

        - Dima






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