his-tagged protein that won't elute

Sergio sergioal at bbm1.ucm.es
Fri Oct 6 14:42:27 EST 2000


We use even higher imidazole concentration, about 100 mM. Actually, even the
lysis and washing buffers include 10 mM imidazole to avoid non specific binding.

just another suggestion, if you are not very worried about the protein amount,
try less "inductive" conditions to avoid the inclusion bodies, it makes the
following purification much easier.

Sergio

Scott Coutts wrote:

> We routinely elute our proteins from IMAC resins (Co or Ni) with 5mM
> Imidazole. It seems to remove most of our proteins, but there have been
> a couple that will not be removed by low pH or imidazole.
>
> >
> > I'm expressing a his-tagged protein in e. coli that forms insoluble
> > inclusions.  I've started trying to clean up the insoluble fraction by
> > using Ni-NTA agarose.  So far in pilot experiments done "batch" style in
> > microfuge tubes (I'm using about 50 ug protein with 25 ul matrix in a
> > volume of 250 ul), I've been able to get nice binding to the Ni matrix, but
> > am unable to elute it.
> >
> > I've been doing this in the presense of 8M urea during all steps.  I get
> > about 80% of my input material to bind at pH 8.0, followed by a pH 6.3
> > wash.  I've tried eluting bound protein with low pH (4.5) and with 100 mM
> > EDTA (pH 8.0) with no luck.  If I boil the post-elution matrix in SDS-PAGE
> > sample buffer, I find the protein.  I haven't tried eluting with imidazole
> > yet.
> >
> > Anybody have any idea what's going on?  One possibility I've thought of is
> > that I'm seeing non specific binding to the agarose beads, but to have it
> > be so much of the protein just seems so unlikely to me.
> >
> > Thanks for any help.
> >
> > Mike
> >
> > Michael L. Sullivan, Ph.D
> >
> > U.S. Dairy Forage Research Center
> > 1925 Linden Drive West
> > Madison WI, 53706
> >
> > (608) 264-5144 Phone
> > (608) 264)-5147 Fax
> >
> > ---






More information about the Methods mailing list