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/
