His6-tagged protein purification

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Feb 24 03:40:41 EST 2000


Angela Manning wrote:

> I am trying to purify a bacterially exressed His6 tagged protein on a
> Probond column under native conditions.  I have purified the protein under
> denaturing conditions, however, the protein is "completely" insoluble after
> denaturation.  So, I want to purify the soluble fraction under native
> conditions.  However, when I do this the protein is contaminated with lots
> of other proteins, even after multiple washes.  I have also tried washing
> in a low concentration (40mM) of imidazole but my protein largely washed
> off under these conditions. Does anyone have any suggestions as to how I
> might purify this protein?  Thank you!
>

Proteins purified from bacteria under native conditions are usually much more
impure than those purified from inclusion bodies. That´s because inclusion
bodies are already quite pure protein
There are, however, ways to get the protein more pure. First try would be
adjusting your extract to 10mM imidazole, 500mM NaCl, and bind to the nickel
material for 2h (no longer). Also, use the lowest possible amount of
ni-agarose to reduce nonspecific binding (we routinely use 0.5ml per 500ml of
bacterial culture).

Hope this helps,
Frank





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