His-tagged protein won't stick to Ni column

jlight at SICKKIDS.ON.CA jlight at SICKKIDS.ON.CA
Sun Nov 17 17:42:33 EST 1996

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.

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