harebrained His-purification question

Emir REMOVEem at unforgettable.com
Fri Dec 7 10:38:26 EST 2001


If you don't want to add the spaces between 6His and the protein sequence,
you might have the only option to denature, purify and then renature your
protein. I understand you have an assay for activity of the protein, so you
should be able to quantitate correctly folded protein and purify it from
misfolded protein. But adding the spacer might be less time consuming. You
may also want to add protease cleavage site after the tag, so that you could
be able to compare the mutant with non-tagged wild-type.
Emir

"Kyle Legate" <legatek at mcmail.cis.mcmaster.ca> wrote in message
news:Pine.SOL.4.33.0112062133070.21368-100000 at mcmail.cis.mcmaster.ca...
> On Thu, 6 Dec 2001, Michael Witty wrote:
>
> > Dear Kyle,
> >          why not dump the French Press?  If you _have_ to use the French
> > Press, can you purify under denaturing condition?  Or should you clone
in
> > some linker codons between your protein and the His tag, to make it
"stick
> > out" some more?
> >               Regards, Mike.
> >
> Thanks for your suggestions. I will be returning to the lysozyme method of
> lysis to see if that corrects the situation. Unfortunately the denaturing
> conditions are no good since I will be carrying out kinetic analysis of
> the protein.
>
> ... . . .  .  .  .    .    .    .     .     .     .      .      .      .
> legatek at mcmaster.ca Kyle Legate            legatek at hotmail.com
>
>    Tower of Tongues:Thursday PM:10:30-11:30 EDT:http://cfmu.mcmaster.ca
>     moon musick:ritual:IDM:experimental(electronica):minimalism:glitch
> .      .      .     .     .     .    .    .    .   .   .   .  .  . . ...
>





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