Make my own resin?

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Mon May 8 03:13:23 EST 2006


Wolfgang Schechinger wrote:

> Hi,
> 
> I'd recommend a crude purification first: Ammonium sulfate
> fractionation, followed by DEAE chromatography or whatever you want to
> get rid of most unwanted stuff (eg proteases!) first. Then, you either
> may load the antibody on some protein A or G column and use it for
> purification (but your AB probably will go off during the elution, too
> and then all your AB will be lost, for a second batch you'll need a new
> load).
> So, better bind your AB covalently to a reactive resin (these tiny
> little pre-packed NHS activated agarose columns from
> Amersham/Pharmacia/GE Healthcare or what their current name is are
> graet for this, but CNBr activated agarose and self packed column will
> do as well. If you want/need to polish your stuff afterwards, that will
> depend on your contaminants. So gel filtration, T7 affinity column, HIC
> or something special else might still be neccessary.

In my experience it is a good idea to start with a affinity step first,
which gives you several 100-fold enrichment of your protein, and then
polish later with steps like ion exchange or size exclusion
chromatography. That way you can use small columns with fine media for
the polishing steps. A typical purification protocol then goes from 50 g
of total protein in 10 l of homogenisate to 200 mg in 20 ml eluate from
the affinity column, the protein may be 90% or so pure already. This
small amount is easily handled on a 5 ml (possibly even 1 ml) HiTrap ion
exchange column for polishing. 

To OP: You can bind an antibody to pre-activated agarose, of which the
CNBr-activated is probably the most well known. Depending on the
affinity and avidity of your antibody you may have to elute your protein
from such a column under harsh, denaturing conditions (e.g. glycine/HCl
pH 4, iodosalycilate). There is no way to predict whether both your
protein of interest and the immobilised antibody will survive this
procedure, only experiments with a small sample can tell. Procedures are
described in

@book{Har-88,
        AUTHOR= {E. Harlow and D. Lane},
        TITLE= {Antibodies --- A Laboratory Manual},
        YEAR= {1988},
        PUBLISHER= {Cold Spring Harbor Laboratory Press},
        ADDRESS= {Cold Spring Harbor},
        LANGUAGE= {engl}
     }

If your protein has an affinity tag, using that for purification can be
an interesting option, in case you have difficulties with the antibody.
If you already have a suitable column, that would be the first route I'd
try. It may not be much cheaper than a home-made column with antibodies,
but you'll save yourself a lot of time, and that is your most valuable
resource. 



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