vladimir vigdorovich vladv at u.washington.edu
Mon Feb 26 03:03:12 EST 2001


I do not add any chelating or reducing agents to the supe. The only things
that I must add are Tris-HCl, NaCl, PMSF, and NaN(3).

On Fri, 23 Feb 2001, Randy wrote:

> 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
> >
>
>
>






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