I have serum containing a custom polyclonal antibody and I'd like to
affinity purify it via interaction with its ligand. I have His-tagged
antigen that I would like to immobilize on a Ni-NTA column. I would, then,
run diluted serum over this column to pull out any antibodies specific to my
antigen, then selectively elute the antibody (ex. with high salt). I know
this is possible and have an idea for a protocol, but I was wondering if any
of you have done this before and have any pointers or protocols that have
worked well for you (i.e. specific buffers, amounts of antibody and antigen
to use, etc).
In article <[Only registered users see links. ].net >, "John Leonard" <[Only registered users see links. ]> wrote:
What do you need the Ab for? If not just for clean Westerns, then
you'd do well doing it longer but the right way.
It's surely been done before but I doubt very much it was ever done
right. With the antigen being bound non-covalently and in a single
purification step, one, IMHO, simply cannot get Ab which can be trusted
in functional studies like inhibition of activity in vitro or
microinjections. Here is how I do it:
1. Purify IgG on Protein A column. Here is the least expensive but
surprisingly well performing resin: [Only registered users see links. ]
(worked better than Pharmacia's HP stuff).
Wash with PBS + detergent, PBS, PBS + 0.5M NaCl, elute with
glycine pH 2.5, immediately neutralize with 1-2 M TRIS.
2. Couple your antigen covalently to an NHS-activated agarose
(e.g., Bio-Rad's Affi Gel 10 or 15, depending on your antigen's pI;
cyanogen bromide activated sepharose is a "standard" but it
leaks considerably more). Make sure to block with ethanolamine
in the end of coupling protocol.
3. Purify your specific Ab on an antigen column. Wash with HBS
(phosphate is not good at this point), 0.5 M NaCl, elute with
3.6M MgCl2 (this is much milder on most antigens than acid
elution, allowing reuse of the antigen column). Do several cycles
of purification until you completely deplete your IgG fraction
(i.e., until nothing binds and elutes from the column). The final
flow through is your control IgG fraction. (And NO, just bought
IgG fraction or even IgG from non-immune or a different rabbit
IS NOT a proper control for functional studies).
IgG is reasonably stable in high MgCl2 but prompt dialysis
into whatever "neutral" buffer is still a good idea.
Just to give some numbers as a reference point from a
latest purification we did in our lab recently:
Two bleeds combined was ~ 24 ml serum from which
about 26 mg of total IgG was purified in the end gave
4.3 mg of antigen-specific polyclonal IgG. (These were early
bleeds; the amount of specific Ab tend to increase toward late
bleeds but this can be bunny-specific).