One week ago I posted this to this newsgroup and have got 3 replys
since then. I hope more users of pGex can give me an E. Mail on their opinions
on these questions, so that I can post a summary next week.
My E. Mail address is BLLIM at molbiol.ox.ac.uk
In article <1993Jul29.202752.1 at molbiol.ox.ac.uk>, bllim at molbiol.ox.ac.uk writes:
> 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
>> 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