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problem kinase domain overexpressed with IPTG
Posted by: biowww (IP Hidden, Regular member, 31)
Date: June 18, 2005 02:00AM
By Akli on 17-Jun-2005
Hallo, I have a problem to guet a kinase domain overexpressed with 0,5 mM IPTG for 2h at 37 C in supernatant. I try different Lysis buffer with socication but without results, the protein always in the pellet! if some one have some recommandations...... [this post was posted as user comment on "Some guidelines for successful gene expression in E. coli" [biowww.net]] Edited 1 times. Last edit at 06/18/05 12:13PM by biowww.
Re: problem kinase domain overexpressed with IPTG
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 24, 2005 12:45PM
I am a little confused. Are you having problems trying to extract protein? You mention changing the lysis buffers. If this is the case, please give me more details about your protocol so I can better help you.
Or having difficulty in dissolving the obtained protein pellet? Some hints to dissolve the pellet: Sonication isn't always the best way to dissolve a protein pellet. Have you tried breaking up the pellet through pipetting? Or incubating the solution, if possible, at a higher temperature to help dissolve the protein? You may trying extending the incubation period at 37C. We have to incubate our samples overnight at 37C to dissolve the pellets.
Re: problem kinase domain overexpressed with IPTG
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: June 24, 2005 12:48PM
you may also try redissolving the protein in 5 to 8 M urea
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