his-tagged protein that won't elute

Achim Recktenwald, PhD ARecktenwald at StressGen.com
Fri Oct 6 16:30:14 EST 2000


a) try to decrease unspecific binding by adding 20 to 50mM imidazole to your
sample

b) try using guanidine hydrochloride as chaotrope; urea and gdn.HCl work
slightly different. Urea unfolds mainly the hydrophobic, non-polar regions,
while gdn.HCl unfolds mainly polar and charged regions.

c) try higher conc. of imidazole to elute; we often have to use in excess of
500mM to elute some of our proteins. In worst case use 6M gdn.HCl + 1M
imidazole to elute strong binding proteins.


Cheers,

Achim


""Michael L. Sullivan"" <mlsulliv at facstaff.wisc.edu> wrote in message
news:v04011700b602a6fcf3cb@[144.92.64.174]...
> 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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