Before loading the Ni-NTA column you should exchange the medium
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 : [EMAIL PROTECTED]
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
