TEA buffer with Ni-NTA column

[Holger Neumann]

Hi Wises Namwat,

you wrote:

> Dear all,
>      Does anybody has experience in purification 6-His-tag protein in
> TEA(Thriethanolamine) buffer with Ni-NTA (phamacia)? As I've tried it
> was eluted in washing step (10mM imidazole). I wonder binding ability is
> quit low because of buffer or my protein's nature.

There is at least one question:
Do you tried to elute native or denatured protein ??

I used Quiagen's Ni-NTA to elute a 60kD kinase under native conditions in a
batch procedure.  I have to admit that it wasn't easy to carry out all steps
under conditions to keep native protein, but I think I managed.
Unfortunately I had a few problems with the eluted protein: it seemed to be
extremly diluted so that all attemps to get quantitation failed.  I tried
using Biorad's solutions as well as western-blot-detection or silver
staining.  Don't know a solution to this problem at the moment.
But kinase-activity assays using [32]P brought best results.

Conclusion:  protein was eluted in native form, but quantitation failed.

I doubt this will actually help you, but what about a little more precise
description of your work.
Perhaps we will find a way.   I also need help with quantitation or even
detection of eluted protein.

Best wishes
Holger
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M.D. student  Holger Neumann
University of Bochum
Germany
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