denaturing buffer for HPLC

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Tue Jun 13 09:38:10 EST 2006


Josmar wrote:

> Dear all,
> I am looking for a denaturing buffer for HPLC. All it should do is
> monomerize a multimeric (don't know if it's actually a dimer, trimer or
> whatnot) protein during gel filtration. The protein has been purified
> by His-tag and Ni-NTA and is reasonably pure. I tried triethylamine but
> it seems to have an adverse effect, i.e. the protein comes off the
> column at the void volume. TEA used to  work fine for me to break scFv
> dimers and trimers. I would like to avoid Stuff like urea or GTC
> because it crystalizes. Any suggestions?

I would avoid ionic denaturants like guanidinium salts if further
purification (IEC) or electrophoresis is desired, but urea is probably
your best bet. This would require finding the smallest concentration
that still monomerises. 

An alternative would be detergents like SDS or CTAB which can solubilise
proteins very well. Note that equilibration of SEC columns with
detergent solutions requires much longer than one would expect, o/n at
low flow rate should do, however. SEC will measure the molecular weight
of the protein/detergent micell under these conditions. Detergent
concentration in the running buffer must be at least cmc to avoid
protein aggregation on the column. If "cmc" and "micell" sound alien to
you, check the review by Helenius (BBA 415 (1975) 29-79).

CTAB is not only effective in solubilising but in many cases even
preserves enzymatic activity. A protein solubilised in CTAB can be
electrophoresed in that detergent (cationic electrophoresis, see Anal.
Biochem. 314 (2003) 70-6).


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