Dear DK
> Lots of people had similar problems.
Thanks for ur interest and advice. I know that probably this denaturing
strategy for my proteins is kind of dead-end:(
> Renaturation on the column is not, in my experience, the most sucessful way of doing it.
Now, in my experience too! My Ni-NTA resign has so many gorgeous
properities in manuals only. I wasn't able to make apropriate sets of
buffers for the protein renaturing which would not destroy the column.
I observed also much weaker binding his tagged protein to the resign in
denaturing conditions.
Try quick 1:100 dilution into 0-0.5 M urea containing (or not) various
GSSG:GSH ratios.
What do you think about this approach? I would free my protein from
inclusion bodies it would make about 5mL of 6M GuHCl solution full of
my protein yet unfolded), make rapid dilution (drop by drop) into 500mL
of appriopriate buffer (eg 20mM MES, about 300-500mM NaCl, some
glicerol - good for avoiding aggregation, isn't it? and minimum
GSSG:GSH level (about 3:0,3mM). Than after some time for refolding I
would flow it through Ni-NTA resign in a column, that wash & elute as
it would be native conditions (using imidasole). What do u think about
it? Is it reasonable?
Waiting for ur opinion
Julio
>> DK
