denaturing buffer for HPLC

Joe Doe None at mail.utexas.edu
Wed Jun 28 19:27:42 EST 2006


In article <1151502137.210567.308880 at x69g2000cwx.googlegroups.com>,
 "Josmar" <josmarlangner at yahoo.de> wrote:

> 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. 

The protein may be eluting in the void volume because the swept volume 
of the unfolded protein is much larger than the folded protein.  How do 
you know it is aggregating?

Analytical ultracentrifugation may be a better way to look at monomer 
dimer equilibria and similar questions.  

Roland


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