affinity purification of Ab using insoluble protein

honanaa at honanaa at
Thu Mar 22 09:46:36 EST 2001

We eluted the proteins (as Ag) from the SDS-PAGE gel, then coupled the protein
to Cyanogen Bromide activated agarose.
The agarose resin worked to purify the antibodies, even the resin appeared blue
(for preparation of Ag, we stained the SDS gel with Comaassie blue, cut gel
slices, eluted the proteins. The protein eluate is blue.)

"Michael L. Sullivan" wrote:

> I've raised a considerable quantitiy of antiserum using protein expressed
> in E. coli.  The protein I used for immunization was SDS gel purified from
> insoluble inclusion body protein.
> Now I would like to affinity purify antibodies.  I've generated a
> considerable amount of insoluble protein (about 50 mg).  What I'm not sure
> is how to go about making an antigen column.  I was thinking perhaps I
> could solubilize the protein in SDS containing buffer, then dialize most of
> it away (I think probably some SDS would remain stuck to the protein
> despite dialysis).  What I'm not sure of is whether this protein
> preparation would then be suitable for something like affigel 10 or 15.
> Has anybody out there been in a similar situation?  Any advice on how to
> proceed?
> Thanks,
> Mike
> Michael L. Sullivan, Ph.D
> U.S. Dairy Forage Research Center
> 1925 Linden Drive West
> Madison WI, 53706
> (608) 264-5144 Phone
> (608) 264-5147 Fax
> ---

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