"Sonicated into solution": really solubilised?

Frank Fürst ffrank at rz.uni-potsdam.de
Fri Mar 5 12:36:15 EST 1999


Hi,

Neil Saunders schrieb:
> 
> Dear all,
> 
> Recently I've over-expressed a small protein using pET22b/BL21DE3 and
> have found it to be relatively insoluble (it fractionates with membranes
> after ultracentrifugation).  On resuspension in Tris buffer plus 0.5%
> Triton-X100 I see small brown globules which are also difficult to
> redissolve.  

Have you tried to dissolve it in guanidinium chloride or urea? Of
course you'll have to find conditions under which it refolds
effectively, but since it is a small protein this should be not so a
difficult task, hopefully.

> be truly solubilised in a strict sense.  I need to be sure that the
> material is at least soluble enough for the next stage (Ni-NTA column),
> so any comments much appreciated.
You can even run the denatured protein on the Ni-column, but of course
its easier to use buffers without denaturant.
Btw, if you use urea, you'll have to be careful of chemical
modification: Don't let it be in that solution too long, and use the
right ionic strength (unfortunately I forgot if it has to be high or
low, but you should be able to look it up)

> email: saunders at bio.vu.nl

Bye, Frank
-- 
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ffrank at rz.uni-potsdam.de
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