On Thu, 16 Nov 1995 07:38:15 GMT,
Richard A. Jefferson <raj at cambia.org.au > wrote:
>We are using His-6 tagging to purify many variants of mutagenized proteins for
>analysis, and are in need of an inexpensive (relatively) method to do so.
>The columns and column resins sold by Qiagen (Ni-NTA), Clontech (TALON),
>Invitrogen (Probond), tend to be very expensive.
>Can someone recommend either an effective 'home-made' version of the Nickel
>chelate methods (and a source for the materials), or give an assessment of the
>relative merits of the above resins, and their re-useability?
My comments are limited to home-made NTA-agarose:
I have successfully synthesized the NTA ligand and coupled it to
agarose (Sepharose CL-4B). It is relatively inexpensive and fairly
easy to do, though a somewhat specialized set up is needed to do the
catalytic hydrogenation deprotection (second step). The
sythesis and coupling using epibromohydrin is described
by Hochuli, Dobeli, and Schacher in J. Chromatography 411: 177-184
(1987). Basically, CBZ-epsilon-lysine (available from Sigma Chemical,
Aldrich Chemical and many other suppliers for US$2.40 to 3.40/g, and
only 3 to 4 g are needed to make enough NTA ligand to couple to 100 ml
of resin) is reacted with one equivalent of bromoacetic acid (very
inexpensive) in 2M NaOH. The protected product is isolated by
crystallization from HCl and is deprotected by catalytic hydrogenation
(on Pd-C). The product is pure after filtering off the catlyst and
drying the residue, though drying is not needed except to determine
yield. Though this sounds a bit intimidating, it is not at all
difficult and is very clean chemistry. If done on the 100 to 300 mmole
scale, you have enough to make a lot of resin (20 mmole/100 ml resin).
The Sepharose is the most expensive component of the preparation.
I have also coupled the ligand to sepharose using epoxy activation
with 1,4-butanediol diglycidyl ether according to Porath and Axen,
Meth. Enz. 44: 19-45. This seems to work just as well for Ni-chelate
West Lafayette, IN
ashendel at aclcb.purdue.edu