affinity purification of Ab using insoluble protein

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Thu Mar 22 09:56:07 EST 2001


Michael L. Sullivan wrote
: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.

If you only need small amounts of purified stuff, then 
purifying on blot strips is the easiest and most convenient
way to go.

Also, did you try renaturation? In many cases about
50% of urea-solubilized protein becomes soluble upon
urea removal by dialysis. This soluble protein might
be the best thing to immobilize for purification. 

Assuming both possibilities above are no go:
SDS is perfectly compatible with NHS chemistry.
I have coupled proteins to Affi-Gel in the presense
of SDS without any problem. After coupling, rigorous
wash of the column will get rid of SDS. The main 
disadvantage of using with such immobilized antigen
for An purification is that the resulting sorbent is
usually quite hydrophobic and gives much higher
amount of non-specific absorbtion of other proteins.
Thus, another purification step (like protein A)
might be necessary. 

        - Dima





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