His-tagged protein won't stick to Ni column

Sai siyer at bmg.bhs.uab.edu
Tue Nov 19 12:44:48 EST 1996


In article <v01530501aeb4fcc17b88@[142.20.35.241]>, jlight at SICKKIDS.ON.CA wrote:

> I've battled for well over a year with getting a His-tagged protein to bind
> to a Ni column.  I've denatured the protein with 8M urea and found that it
> passes right through the column. My protein is about 60 kD, but I've had
> the same problems with a 15 kD piece of the same protein.  How sensitive is
> His-tag binding to pH? (i.e. will 0.1 or 0.2 pH units matter?).  I usually
> use pH 8 - 8.5 (at r.t.)  without being too fussy.  I've tried about every
> His-tagged vector on the market.  Any advice is most appreciated.



i've done many his-tagged fusion protein purifications.  i use the
pharmacia  hitrap chelating columns in conjunction with novagen's pET
vector system.  my binding conditions are in a high conc. of imidazole
(100 mM) and i elute it off with 500 mM imidazole.  my yield is pretty
damn good (15-20 mg average per litre of culture) and my protein is pretty
damn clean (i.e no contaminants)
.  i do this under native conditions.  denaturing conditions require that
your imidazole binding conc. be really low.  so the question to ask would
be what conc. of imidazole are you binding in?  if i were you, then i
wouldnt go any higher than 10 to 20 mM imidazole.  at denatureing
cnditions, the his-tag is exposed much more than it is in native
conditions.  this is why it takes less imidazole to bind to the nickel
column.  also, how much nickel are ou charging your column with?  i charge
mine with 100 mM NiSO4.  alternatively, if you are using the hi-trap
columns from pharmacia, you might wanna try charging with copper or zinc
which have the same valence as nickel and should work just as well (the
pharmacia manual for the hitrap columns mention ths as well).  the buffer
i use is a phosphate buffer at pH 7.8.  although using buffers at pH 8
should also work fine.  that's all i can think of.  hope this helps...good
luck...cheers...

-- 
Sai Iyer
siyer at bmg.bhs.uab.edu
graduate student, Dept of Biochemistry and Molecular Genetics
University of Alabama at Birmingham...



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