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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: jbosch {- at -} U {- dot -} WASHINGTON {- dot -} EDU Date: 2007-02-28 > 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 CCP4bb navigationCCP4bb <-- 2007 <-- February 2007 <-- 28 February 2007 |
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