denaturing buffer for HPLC

Josmar josmarlangner at yahoo.de
Wed Jun 28 08:42:17 EST 2006


OK folks,
thanks for your opinions so far. I will give a little more details.
Don't be angry because I still do not name the protein. Anyway, we have
a wt and a mutant form. The mutant is supposed be no longer able to
dimerize. We did gel filtrations with both on a Superdex 200 and saw
one peak for the mutant and a similar peak with a little bit of a
shoulder towards bigger stuff for the wt. So far so good. To confirm
the shoulder is indeed a dimer/multimer we tried denaturing running
conditions. 100mM TEA and 7M urea. Both causes the protein wt and
mutant to aggregate and to elute at the column's void volume. Of course
one could collect fractions and analyze them by other means. But I
would still like to understand what is going on here. As far as running
the column under detergent is concerned, instrument time is very
limited and Engelbert warned us about excessive equilibration times.
Even more important, if micells are the entities that are analyzed
under detergent and there is a size distribution among them, I would
expect peaks to broaden.
Cheers
Josmar


DK wrote:

> Well, it aggregates strongly enough for 7M urea non being able
> to disrupt the interactions. Happens... There are SDS-resistant
> protein complexes. I'd try next very alkaline conditions.
> Say, 150 mM NaOH. This should be OK to use with all except
> silica-based gel-filtration columns.
>
> Also, if you don't care about protein denaturation - why not simply
> run an SDS gel instead of HPLC? Preparative gels are not such
> a big deal, really. 
> 
> DK



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