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.
-mdsr-
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