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Re: [ccp4bb] Cannot running NTA to purify the protein having His-tag? |
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CCP4bb navigationCCP4bb <-- 2007 <-- February 2007 <-- 28 February 2007Subject: Re: Cannot running NTA to purify the protein having His-tag? From: jmdias {- at -} SCRIPPS {- dot -} EDU Date: 2007-02-28 buffer (some media have histidine which will compete with your protein) to a more suitable buffer, like your loading buffer (Buffer A is 50mM phosphate buffer pH 7.5 and 300mM NaCl is OK). If your His-tag is not accessible, then follow the other suggestions or try different tags in different places. Good luck, Joao On Feb 28, 2007, at 11:21 AM, Alex Berndt wrote: > sometimes the insect cell medium intereferes (for whatever reasons) > with nta purifications when they ar employed as a first step in the > purification scheme. i experienced that occasionally. this can > easily be circumvented by doing an ion exchange step beforehand! > alternatively you might want to introduce a linker between your > protein and the his-tag or create a 8xhis or 10xhis tag to enhance > bing to the nta matrix. make sure you wash your cells from residual > medium before you freeze your pellets. > > alex > > On 28 Feb 2007, at 19:18, Juergen Bosch wrote: > >> Ngo Duc Tri wrote: >> >>> Dear CCP4 users, >>> >>> I'm purifying a kind of protease having His-tag. The protein is >>> expressed in insect cells and broken by sonication. >>> I used NTA resin to purify this protein. >>> Buffer A is 50mM phosphate buffer pH 7.5 and 300mM NaCl. Buffer B >>> is 50mM phosphate buffer pH 7.5, 300 mM NaCl and 300 mM Imidazole. >>> However, all proteins cannot bind to NTA resin. My protein is >>> eluted in Flow-through. I also check the NTA resin with the >>> control His-tag. The western blot also shows that my protein has >>> His-tag. >>> >>> Do you have any ideas about my problem? I'm really appreciate all >>> of your advices how to solve this. Thank you very much! >>> >>> My best regards, >>> TriNgo >>> Sungkyunkwan University >>> >> You His tag is most likely inaccessible, can you easily change the >> tag from e.g the N-terminus to the C-terminus ? Or if you have a >> structural homolog you could add the His tag into a loop, which is >> exposed. >> >> Alternatively you can purify your protein under denaturing >> conditions using 8 M urea and refold it if you dare :-) >> >> Juergen >> >> -- >> Jürgen Bosch >> University of Washington >> Dept. of Biochemistry, K-426 >> 1705 NE Pacific Street >> Seattle, WA 98195 >> Box 357742 >> Phone: +1-206-616-4510 >> FAX: +1-206-685-7002 > > --- > Alex Berndt > MRC Laboratory of Molecular Biology > Hills Road > Cambridge CB2 2QH > UK > > mail : aberndt@mrc-lmb.cam.ac.uk > phone : +44 (0)1223 402113 > --- > > > Joao M. Dias The Scripps Research Institute 10550 North Torrey Pines Rd. IMM-2 La Jolla, CA 92037 USA tel (858)784-8925 CCP4bb navigationCCP4bb <-- 2007 <-- February 2007 <-- 28 February 2007 |
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