How imidazole can be washed off the Ni-NTA agarose beads - reuse of Ni-NTA agarose beads for His protein (Jan/12/2005 )

Can someone guide me on how to get rid of imidazole adsorbed to Ni-NTA agarose beads so they might be reused. Thanks in advance.

Download this PDF document :
http://www1.qiagen.com/HB/QIAexpressionist

You will find lots of informations about NiNTA purification system. And a protocol for Ni-NTA regeneration...

You can wash your resin with 0.5N NaOH 30min for rapid regeneration. And use the same resin for the same protein purification...

For information :

Handling :

Ni-NTA matrices are stable under a wide variety of conditions and need not be refrigerated,
except to inhibit growth of microorganisms for long-term storage. After use they should be
washed for 30 minutes with 0.5M NaOH. Ni-NTA matrices should be stored in
30% ethanol to inhibit microbial growth. The matrix can be stored for up to one week in
any of the denaturing buffers.

Reuse of Ni-NTA Resin

The reuse of Ni-NTA resin depends on the nature of the sample and should only be performed
with identical recombinant proteins. Based on the experience of Hoffmann-La Roche Ltd.
(Basel, Switzerland), who have purified more than 100 different proteins on Ni-NTA resin,
we recommend a maximum of 5 runs per column.


If the Ni-NTA Agarose changes from light blue to brownish-gray, the following regeneration
procedure is recommended.
Procedure:
1. Wash the column with 2 volumes of Regeneration Buffer (6 M GuHCl, 0.2 M acetic
acid).
2. Wash the column with 5 volumes of H2O.
3. Wash the column with 3 volumes of 2% SDS.
4. Wash the column with 1 volume of 25% EtOH.
5. Wash the column with 1 volume of 50% EtOH.
6. Wash the column with 1 volume of 75% EtOH.
7. Wash the column with 5 volumes of 100% EtOH.
8. Wash the column with 1 volume of 75% EtOH.
9. Wash the column with 1 volume of 50% EtOH.
10. Wash the column with 1 volume of 25% EtOH.
11. Wash the column with 1 volume of H2O.
12. Wash the column with 5 volumes of 100 mM EDTA, pH 8.0.
13. Wash the column with H2O.
14. Recharge the column with 2 volumes of 100 mM NiSO4.
15. Wash the column with 2 volumes of H2O.
16. Wash the column with 2 volumes of Regeneration Buffer.
17. Equilibrate with 2 volumes of a suitable buffer (e.g., Buffer A or .

Good luck.
David.

Hi, so in my protocol it says that regeneration should be performed if the Ni-NTA agarose turns from light blue to brownish-gray.

Am I right to assume that if the agarose is still light blue, that I just need to wash my resin with 0.5N NaOH 30min for rapid regeneration, and then use it again?

Thanks for your help!