(none)

To: Nikolai troianovsk_s at MSDISK.WUSTL.EDU
Sun Mar 26 21:48:44 EST 1995


>Date:   23-MAR-1995 19:17
>From: sherif at ORCHID.UCSC.EDU (Sherif Abouelela)
>Description: (none)
>
>Hi to all:
> 
>        Does anybody know an easy way to reduce non specific binding of
>proteins to the Ni-NTA spin column. I am trying to purify a 54,000
>dalton  protein from yeast using a six his tag. While I can see my protein
>using immunoblotting tons of other stuff also stick to the column [as judge by
>staining]. Any ideas or tricks.
> 
>Sherif Abouelela


        Well, there are a couple of things you could do about:
i) play with pH, (bring it down to 5.9);
ii) add some detergents like Triton X100; NP40, Tween 20 up to 0.5 %;
iii) add beta-ME up to 10 mM;
iv) add Imidazole up to 40 mM;

Then elute either by lowering pH to 4.5, or increasing imidazole
concentration to 80 mM, or add 250 mM EDTA to any buffer.

Nikolai.





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