his-tagged protein that won't elute

Scott Coutts scott.coutts at med.monash.edu.au
Fri Oct 6 05:05:19 EST 2000


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
> 
> ---






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