His tagging and 'cheaper' Nickel columns

Nikolai Chitaev nikolaic at VISAR.WUSTL.EDU
Thu Nov 16 12:44:58 EST 1995


> To: methods-and-reagents at net.bio.net
> From: raj at cambia.org.au (Richard A. Jefferson)
> Subject: His tagging and 'cheaper' Nickel columns
> Message-ID: <raj.29.30AAEA67 at cambia.org.au>
> i all,
> 
> 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?
> 
> Richard
> 
The re-using of Ni-NTA resin is recommended with the SAME protein a max. of 3-5 runs
per column.
However, the used resin could be recharged anew.
Procedure:
1.Wash used resin with 2 vol. water;
2. --"--  3 vol 2%SDS;
3. --"--  1 vol 25% EtOH;
4. --"--  1 vol 50% EtOH;
5. --"--  1 vol 75% EtOH;
6. --"--  5 vol 100% EtOH;
7. repeate step 5;
8. --"--   step 4;
9. --"--   step 3;
10. Wash with 1 vol of water;
11. Wash with 5 vol of 0.5 M EDTA, pH 8.0;
12. Wash with 2 vol of water;
Optional (Steps 11 and 12 could be repeated 3 times)
13. Recharge the resin with 5 vol of 0.1M NiSO4.
14. Wash with 5 vol of water.

P.S. Never did it myself.
good luck
Nikolai
http://128.252.119.253






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