His-tagged protein won't stick to Ni column

[blah at facstaff.wisc.edu]

In article <siyer-1911961146180001 at bmg147.cmc.uab.edu>,
   siyer at bmg.bhs.uab.edu (Sai) wrote:
->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)

Just look at one of these lucky individuals... FYI, not many his-tagged
proteins behave this way... 


->..  i do this under native conditions.  denaturing conditions require 
that
->your imidazole binding conc. be really low.  

??? 
How come? 

->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.  

???

The more 6His is exposed, the stronger is binding, the higher imidazole
is required to elute (in other words, the higher imidazole conc. could be
used under binding conditions).

->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).  

Yeah, and pick up all those proteins that bind to chelated Zn and Cu, 
but do not bind to Ni (FYI, Ni is much more specific for His, while other
divalents bind other aromatics as well). 

- Dima