harebrained His-purification question

Michael Witty mw132 at mole.bio.cam.ac.uk
Thu Dec 6 17:15:13 EST 2001


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.

. . . or move the tag to the C terminus?

On Thu, 6 Dec 2001, Kyle Legate wrote:

>
> I'm having serious difficulty getting a His-tagged protein I have
> expressed to bind to Ni-NTA agarose. The wild type protein purifies very
> well in 50mM NaHPO4, 500mM NaCl, 5mM MgCl2, 10% glycerol, french press
> method of cell lysis. It elutes in 50mM imidazole, so the binding is not
> that tight. However, a point mutant I have made will not bind
> under those conditions, nor by reducing the NaCl to 300mM, switching the
> buffer to 40mM Tris-Ac, pH 7.8, or adding 10mM beta-ME, all of which
> should be tolerated by the resin. Looking back through my notes I notice
> that pilot experiments worked reasonably well when I lysed the cells with
> lysozyme + 13% sucrose, but have tanked since I switched to the French
> press. Does anyone have any recommendations to help me escape this cycle
> of misery?
>
> ... . . .  .  .  .    .    .    .     .     .     .      .      .      .
> 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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