Cleaning up primary antibodies??
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.
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