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bllim at molbiol.ox.ac.uk bllim at molbiol.ox.ac.uk
Thu Jul 29 14:27:52 EST 1993


Dear All:

	I am a pGex-2T user and I want to ask two questions. I will post a 
summary on these questions later.

1. Do you add protease inhibitor(s) in your E. coli lysis buffer? I used 10 mM 
EDTA and 0.1mM Benzamidine but still got quite a lot of degradation products 
even though I did all the steps in cold room or on ice.(centrifuge, 
resuspend, sonication, affinity column.... I think the cleavage is post-lysis 
since I could see intact protein band in total cell lysate but it was degraded 
afterwards.

2.How do you wash your GST agarose before eluting your fusion protein by 
Glutathione? Can I use up to 0.5 NaCl or up to 1.0 M NaCl in my wash buffer 
to remove non-specific proteins before elution? I could see other non-specific 
proteins in SDS-PAGE after elution.

Thanks in advance.
Pls post to my account if possible so that I can summarize easily.

Wallace Lim
BLLIM at molbiol.ox.ac.uk



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