his tagged protein

[Knut Tomas Dalen]

In article <SS32-1505971738280001 at mac65.msb.le.ac.uk>, SS32 at le.ac.uk says...
>
>hello
>i'm trying to purify a HIS TAGGED protein on nickel-NTA RESIN, from
>qiagen. i'm having very little binding and very poor recovery . i need to
>purify under native conditions , does anyone have any suggestions please.
>please reply to ss32 at le.ac.uk
>thanks
>shital
>leicester university

Hei

I’m purifying a His-tag protein myself, with a FPLC form Pharmacia.
I am amplifying 1 ml radioactivity ( 100.000 cpm ) marked proteins with only 
the cell medium as a source. The column are HiTrap from Pharmacia.

I use this two buffers:
Buffer A :  20 mM phosphate 500 mM NaCl    pH 7.4
Buffer B:   20 mM phosphate 500 mM NaCl 100 mM imidazole  pH 7.4

The FPLC are programmed to add a gradient of buffer B.
Most of the material goes through with 4 ml of pure buffer A.
Then the concentration of B are set to 10 % , and a new top loosely bind 
protein are eluted.
The His-Tag bound protein elutes around 20 -50 % buffer B.

The sample must be in buffer A. I tried ones with samples containing Triton, 
but that did not work well.
Another protein eluted together with the His-tag protein.

Mail me if you need more information.
Knutd at biokjemi.uio.no

Knut Tomas Dalen