Randy rldurren at bchserver.bch.ncsu.edu
Fri Feb 23 09:48:56 EST 2001


Mr. Vigdorovich,
Do you have something like DTT or EDTA in your buffer. I use Tris-HCl (pH
7.9) regularly on my His-bind column, w/o problems.
Randy

"Roger Murphy" <roger.murphy at ludwig.edu.au> wrote in message
news:3A95F0BD.B679639B at ludwig.edu.au...
> Tertiary amines such as Tris can strip metal ions from the column - change
> your buffers.
>
> Cheers,
> Roger
>
> vladimir vigdorovich wrote:
>
> > Hi, I am trying to optimize a purification procedure of a His-tagged
> > protein derived from Hi5 cells using Talon resin. When I apply
clarified,
> > pH-adjusted (to ~8.0), buffered (supplemented with 20 mM Tris-HCl) supe
to
> > the column, it slowly changes color to whitish-pink and the flowthrough
> > darkens. Some protein binds but I suspect that much ends up in
> > flowthrough.
> > Can anyone verify my suspicions that Co2+ is being leeched and/or
suggest
> > a remedy?
> > Thanks,
> >
> > Vlad
> > vladv at u.washington.edu
>







More information about the Proteins mailing list