affinity purification of Ab using insoluble protein

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Wed Mar 21 14:47:21 EST 2001


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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