Ion Exchange Chro/HPLC with 6M Guanidine or Some Detergent?

John Philo jphilo at
Tue Mar 11 11:59:05 EST 1997

newera at wrote:
> Do not 6M ganidine HCl, 8M urea or some zwitter-ionic detergent make any difference to cation exchange chromatography or affinity chromatography?
> My protein tends to be aggregated during some purification steps such as dialysis or concentration with a Centriprep. In the midst of misfortune, my protein has no cysteines and seems to be easily renatured; so I plan to add 6M guanidine HCl/8M urea and 0.5~2% or more zwitter-ionic detergents such as CHAPS, sarkosyl to my current buffer system(20mM Na Phosphate, pH 6.8).
> I am deeply concerned that they severely interfere with my protein's binding ability to BioRex70 cation exchange resin, BlueSepharose affinity chromatography resin, to which my protein seems to quite tightly bind since it begins to elute at around 0.5M NaCl, and C18 reverse phase HPLC.

You will certainly not be able to use ion exchange chromatography in 6M
GdnHCl!  The extrememly high ionic strength from that addition would
completely shield the electrostatic interactions of proteins with the
column and thus prevent binding/separation.

It is possible to use ion exchange with (non-ionic) urea, although I
don't think I have ever heard of anyone doing it with urea
concentrations that high.

Usually when people add urea to help minimize aggregation they would not
add enough to completely denature the protein of interest, but just
enough to help keep things soluble (perhaps 1 M).

Those amounts of either GdnHCl or urea also would be incompatible with
dye-binding and reverse phase chromatography.

Also, one problem with ion exchange for proteins which tend to aggregate
is that typically your protein all sticks near the top of the column and
thus is very highly concentrated and prone to aggregate.  Thus you may
want to consider chromatography under conditions where the impurities
will bind but your protein will flow through.

Also, you stated that you believe your protein re-folds easily.  Are you
sure this is true?  Often aggregation of recombinant proteins,
especially from E. coli (I am guessing that is what you have) is really
due to the presence of some mis-folded protein.

Good luck,

John Philo, Protein Chemistry, Amgen
*** Disclaimer: These are the opinions of the poster not Amgen Inc.***

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