Column for pET system??

Curt Ashendel ashendel at aclcb.purdue.edu
Tue Jan 24 15:12:38 EST 1995


On Sun, 22 Jan 1995 08:18:52 GMT, 
bllim at hkucc1.hku.hk  <bllim at hkucc1.hku.hk> wrote:

>        I am going to express some His-Tagged fusion protein and need to 
>purchase the affinity matrix for protein purification. Since Novagen is not
>available in HK and it may take several months for ordering, I wonder if other
>companys do supply the same products. Alternatively, can I make the affinity
>matrix by myself? 

In addition to Novagen, Quiagen and Invitrogen, an IDA (imminodiacetic 
acid) agarose is available from Sigma.  NTA-agarose can be synthesized  (I 
have done so) for a small fraction of the cost of the resin if you plan to 
use it a lot.  The chelator that you need to link to the agarose is not 
sold commercially and must be synthesized using two aqueous reactions. The 
starting materials are bromoacetic acid and Z- (or CBZ-) epsilon-N-lysine, 
which is sold widely (Sigma, etc). The synthesis takes only a day to do, 
with coupling done overnight.

The reference for both synthesis and coupling (to epibromohydrin activated 
agarose) is:

E. Hochuli, H. Dobeli, and A. Schacher 
J. Chromatography  411:  177-184, 1987.

This works OK, but I am still trying to increase the capacity of the 
column to be more like glutathione agarose.  

Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu



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