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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: aberndt {- at -} MRC-LMB {- dot -} CAM {- dot -} AC {- dot -} UK Date: 2007-02-28 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 --- CCP4bb navigationCCP4bb <-- 2007 <-- February 2007 <-- 28 February 2007 |
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