Dear Neil!
I would either filtrate your sonicated suspension through 0.1um
(0.22um) filter or ultracentrifuge it at 400,000g for 1 hr. before loading
on the column ( I usually use centrifugation). Pay attention to any
possible pellet after spinning (the pellet can be transparent). Various
aggregates and unsolubilized membrane vesicles will form this pellet. Make
sure that you check protein concentration and protein contents before and
after centrifugation (filtration): SDS-PAGE and any colorimetric
quantitative protein assay will work.
Good luck,
Lena.
Neil Saunders wrote:
> 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. When I briefly sonicate the suspension, these globules
> disappear, the solution becomes brown and the protein runs nicely on
> SDS-PAGE. The question is, does sonication really bring material into
> solution? I can conceive of a situation where particulates would be
> broken up into small enough pieces to be suspended and not
> precipitatable by normal centrifugation, and they would also be
> accessible to and dissolve OK in SDS-PAGE sample buffer, but might not
> 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.
>> Neil Saunders
>> --
> Department of Molecular Cell Physiology,
> Faculty of Biology,
> Vrije Universiteit,
> De Boelelaan 1087,
> 1081 HV, Amsterdam
> The Netherlands
>> phone: +31 20 4447194
> email: saunders at bio.vu.nl> WWW: http://members.xoom.com/paracoccus/
