IMAC, reply to Dima Klenchin
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Mon Dec 4 12:06:12 EST 2000
marese at ircc.unito.it (Marco Arese) wrote:
:Thanks for your suggestions.
:The trouble with my IMAC is that my protein binds to the Nickel resin
:only when is denatured. So I use urea 8M as a denaturant, then I use an
:"on column" refolding from 8M to 0 urea followed by an elution with
:0-500 mM imidazole, "my" protein elutes from 200 to 400 mM (very broad,
:I understand, but some fractions are 99% pure by silver stain)
Silver stain? What kind of expression system do you use that
you have to use silver to see your protein? If you have that low levels
of expression, then your best bet would be get overexpression
working nicely (in inclusion bodies if E.coli; since your protein
renatures, then just make a lot of incl. bodies, purify them
(already ~70% pure protein), and purify further by IMAC. If your
protein elutes at >200 mM, then I would wash the column
extensively (20-50 column volumes) in urea+100 mM imidazole,
then elute either by gradient or step wise with 100 mM
increment. Alternatively, you can try low pH elution (which
IMO always produces durtier preps but might work better
in your case).
:trouble is that the elution does not follow a reproducible pattern, so
:that in many experiments the protein elutes aspecifically with many
:others around 250 mM imidazole.
"Many others around 250 mM" sounds extremely strange. In my
experience, when the conditions are right (high salt, pH 6.5 - 7.5, etc),
there are no proteins that stick specifically at >~ 100 mM imidazole,
and certainly not at 250.
I obviously loose control over some
:critical parameters of the chromatography, especially when I try to
:scale up the amount of total protein loaded (I use a 1 ml Hitrap
:chelating column).I also tried to elute my protein under denaturing
:conditions, but the results weren't any better than before.
Perhaps try another sorbent. Hitrap chelating is highly crosslinked
agarose that exhibit somewhat higher non-specific binding. It also
has IDA linked to it, which is not as good a chelator as NTA. Have
you tried Ni-NTA from Qiagen?
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