TEA buffer with Ni-NTA column
holger.neumann at ruhr-uni-bochum.de
Wed Feb 24 17:26:08 EST 1999
Hi Wises Namwat,
> 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 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.
M.D. student Holger Neumann
University of Bochum
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