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Survey on pGex vector

bllim at molbiol.ox.ac.uk bllim at molbiol.ox.ac.uk
Wed Aug 4 13:26:59 EST 1993


Dear All:

	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 
> 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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