help! purifying polyclonals (fwd)
haoxiao at cc.UManitoba.CA
Wed Oct 26 22:38:30 EST 1994
BARKER, SHARON L. writes:
> From BIOSCI-REQUEST at net.bio.net Wed Oct 26 20:36:58 1994
> To: immunology at net.bio.net
> From: "BARKER, SHARON L." <BLY3000 at MUSICB.MCGILL.CA>
> Subject: help! purifying polyclonals
> Date: 26 OCT 94 20:57:38 EST
> Sender: usenet at MUSICB.MCGILL.CA
> Message-ID: <26OCT94.22637482.0262 at VM1.MCGILL.CA>
> NNTP-Posting-Host: vm1.mcgill.ca
> I'm an MSc student trying to purify a subset of monospecific
> human antibodies from polyclonal rabbit serum. I have tried
> binding my ligand to Affi-Gel 10 but don't get good binding.
> I have also tried binding the ligand to nitrocellulose and
> eluting bound antibodies, but i can't seem to get enough to
> use for immunofluorescence. Short of using up every drop of
> antigen and antibody to get enough for 1 slide are there any
> other good methods and/or optimization tips? Thanks.
> Sharon Barker Biology Dept. McGill Univ.
before i suggest anything, i would like to know 1) why human antibodies
mixed with rabbit anti-serum? 2) what is (are) the immobilized ligan (protein,
small hapten eg.) and what is the chemistry for immobilization? 3)
what was your eluting buffers? 4) how did you confirm that the Ab's were
actually bound to the ligand (if it existed)?
you even could bombard the avidin out of the biotinylated column, so it is
nothing for Ag-Ab complex.
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