gal-1 at bones
Tue May 9 08:55:00 EST 1995
On Mon, 8 May 1995, Christer Ericsson wrote:
> In article <3ohqli$795 at worak.kaist.ac.kr>, dykim at bioneer.kaist.ac.kr
> (Dooyeon Kim) wrote:
> > Instead of using cDNA subtraction or 2D PAGE analysis, I will use immunosubt
> > ractive methods. The princeple of this method is producing the polyclonal
> > antibodies against whole protein mixture of tissue or cultured cell and select
> > ing the antibodies against newly expressed protein by passing the antiserum
> > against the protein(antigen) column of control tissue of cell line.
> After immuno
> > subtraction, the newly expressed protein will be easily purified by immuno
> > precipitation and detected on SDS PAGE gels. Dose anybody advice me about this
> > idea? Any Comments will be greatly appreciated. Thanks for reading
> I have been toying with similar ideas for some time, but never got around
> to trying it. I suspect one may have problems with some (many?) proteins
> not being very immunogenic in a complex mixture (due to immunodominance or
> whatever) and so after spending time and money on raising and depleting
> antiserum, you may end up not being able to purify your protein.
> Is there anybody out there who actually tried, and is willing to share
> his/her experience?
> Christer Ericsson, Dept. of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, S-171 77 Stockholm, Sweden.
I tried something like this years ago; I suspect lots of people have and
the reason that you don't read about it is that it doesn't work. The
problems are 1) getting absolutely clean subtractive absorption is
practically extremely difficult if not impossible and 2) the small
proportion of differentially reactive antibodies in the serum can not be
once the absorption is done, which means that repeated enrichments can
not be performed as can be done with subtractive hybridization.
If you want to go this route, then I would suggest looking into
phage display libraries for producing your antibody repertoire. At least
there are published reports of successful cycles of enrichment and
amplification. THe only problem there is that this approach is still
being developed, so you can't find a good cookbook method with well
worked out optimization protocols yet.
Warren Gallin wgallin at gpu.srv.ualberta.ca
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