his-tagged protein that won't elute

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Oct 5 16:55:39 EST 2000


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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