Making Nickel resin??

Curt Ashendel ashendel at
Thu Feb 27 11:24:01 EST 1997

On Thu, 27 Feb 1997 09:21:36 +1100, Stephen W Doughty   
<swd at> posted to bionet.molbio.methds-reagnts:

>I interested in making my own metal affinity resin to purify a (HIS)6
>tagged protein, can anyone tell me where I can get a protocol from to
>do this?

Last November someone asked the same question. Here is the same reply, 
with a correction and some comments added that came out of the 
discussion of the subject. Some of these postings may be in the 
methds-reagnts database (see the FAQ for how to access the database). 

First of all, two types of Ni-chelator columns have been used for 
IMAC (immobilized metal affinity chromatography): IDA-sepharose 
(imidodiacetic acid) and NTA-sepharose (nitrilotriacetic acid). When 
coupled to the resin, the IDA chelator has two carboxylates for 
chelating the Nickel ions, while the NTA has three. The 
research literature indicates that the NTA resins bind the metal ion 
more strongly with less blead off. I have not seen a direct comparison 
of the two columns for their loading capacity or purification 
efficiency when used for IMAC of proteins, but claims of superiority 
of NTA-sepharose are made by some suppliers that sell that resin. 

Sigma sells IDA-sepharose if you want to try it. They do not sell 

IDA and NTA, while commercially available, cannot be coupled to the 
resin.  To couple the chelator to the resin, one needs to use the 
blocked form of the chelator. The chelator must have a functionakl 
group that allows the coupling to occur. For NTA-sepharose, this 
compound is not NTA, but N-(5-amino-1-carboxypentyl)iminodiacetic 
acid. Last I checked, this compound is not available commercially and 
must be synthesized in two fairly easy steps.  This is done by adding 
two acetic acid groups to a blocked lysine followed by deprotecting.

The synthesis of the NTA lignad and its coupling to Sepharose is  
relatively inexpensive and fairly easy to do, though a somewhat 
specialized set up is needed to do the catalytic hydrogenation 
deprotection (second step). For this I had access to such a set-up in 
the lab of a departmental colleague. 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 two 
equivalents 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 

There are some more detailed tips I can send on request about how do 
do the hydrogenation or the epoxy activation if you want to try to do 
this yourself. 

Curt Ashendel
Purdue University, West Lafayette, IN
ashendel at

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