Osmotic shock

N490047 at UNIVSCVM.CSD.SCAROLINA.EDU N490047 at UNIVSCVM.CSD.SCAROLINA.EDU
Mon Jul 3 08:27:15 EST 1995


In article <3sl6cr$jca at nuscc.nus.sg>
medp4003 at leonis.nus.sg (Farid John Ghadessy) writes:
 
>
>Hello all,
>
>I've been trying to isolate a protein that I'm expressing in the
>periplasm  using osmotic shock (Neu and Heppel). The problem is that I
>can't see any bands on an SDS gel after shocking, suggesting I'm doing
>something wrong. I'm only inducing a 10ml culture to test things before I
>scale up but don't seem to be able to liberate ANY proteins from the
>periplasm. My questions.....are certain cell lines less "shockable"
>and/or should I scale up to really see anything.
>
>------------------------------------------------------------------------------
Are you sure your expression system is working?  I have had variable results
using osmotic shock--I don't know if it is my incompetence though.  A more re-
producible method is to make spheroplasts with lysozyme.  There is a technique
for this in _CPMB_ under RNA isolation from G- bacteria.  However, if your
expression system is working well, you should be able to detect your protein
in a crude lysis on SDS-PAGE.  Let me know if you need more info.
 
--Tom
 



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