purification of His-tagged proteins

Wulf Dirk Leuschner wdl at gen.mpib-tuebingen.mpg.de
Tue Dec 10 17:01:15 EST 1996


Volker Blaschke <vblasch at gwdg.de> wrote:

>Who has managed to perform a purification of a His-tagged protein from 
>mammalien cells ( in this case HEK 293 cells ) with a nickel -chelate 
>column ?
>Was it a special kit that you have taken ?
>Which detergens have you used to solubilize the protein ? (in case of a
>membrane-standing protein )
>I would be delighted to get some information about it !
>My E-mail:  uboeer at gwdg.de

Hi Volker,

I am trying to purify His-tagged membrane proteins expressed by
COS7-cells. After solubilizing them using 1 % (w/v) CHAPS, 0.5 M NaCl
in PBS (pH 7.1) with some protease inhibitors, I incubate the
solubilized proteins with 25 ul Beads (50 ul 50 % slurry) for 2 h at 4
°C in a batch procedure.
However,  I have not yet been able to get specific binding to
the Ni-NTA-agarose higher than 10 - 20 %. I've still got the problem
of unspecific binding to the material. I tried using 10 mM imidazole
in the binding buffer: no effect. I tried washing the beads with
mounting concentrations of imidazole (up to 20 mM): no apparent
effect. I have talked several times with the technical service of
Qiagen, but they did not really come up with new ideas.
There are still some experiments I haven't done yet like using
glycerol (about 10 %)  in the binding and washing buffer, or blocking
the column material with BSA before adding the cell extract. Another
possibility is to use a higher pH (a pH of 8.0 is recommended which
might present problems with some experiments). Qiagen says they used
pH 7.5 or lower before and they had slightly lower binding compared to
'normal' conditions with a pH of 8.0, but it never was as bad as in my
experiments. In fact, some proteins do elute at pH 6.7, so maybe
increasing the pH indeed does the trick in my case...

Maybe you have more luck, and I would really appreciate 'new'
information on how optimal binding with detergent solubilized membrane
proteins with the Ni-NTA-agarose can be achieved.

WDL.

e-mail:	wdl at gen.mpib-tuebingen.mpg.de




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