Crm at 16:15, 7 February 2018

2018-02-07T16:15:08Z

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1. If column is very brownish-gray, wash with 3 column volume (CV) water, 0.5 M NaOH then water.

2. If column is still very dirty, wash the column with 2 volumes of GnHCL Regeneration Buffer (6 M GuHCl, 0.2 M acetic acid), then several CV of water.

3. If column need to be washed with SDS:  you can wash Ni column with 5 volumes of H2O, then 3 volumes of 2% SDS and 5 CV of water.

=== Routine regeneration: ===

4. Wash the column with 2 CV of water,  then  1 CV of 100 mM EDTA/500 M NaCl, <s>pH 8.0</s>.

5. Wash the column with 2 CV of H2O.

6. Optionally, wash column with 0.1 N NaOH/1 M NaCl.

7. Recharge the column with 1 column volume of 100 mM NiSO4, or NiCl2.

8. Wash the column with 10 column volumes of H2O.

9. Equilibrate with 3 volumes of a suitable buffer (''e.g., ''Buffer A).

'''GnHCL Regeneration Buffer (1 L)''' 6M GnHCL 573 g of Guanidine 0.2M Acetic Acid 11.4 mL Acetic Acid This buffer must be 0.45 um filtered to remove particulate before use.

'''Deep Cleaning'''

The Ni2+-stripped column can be cleaned by the following methods;

 • Remove ionically bound proteins by washing the column with several column volumes of 1.5 M NaCl; then wash the column with approximately 10 column volumes of distilled water.

 • Remove precipitated proteins, hydrophobically bound proteins, and lipoproteins by washing the column with 1 M NaOH, contact time usually 1–2 hours (12 hours or more for endotoxin removal). Then wash the column with approximately 10 column volumes of binding buffer, followed by 5–10 column volumes of distilled water.

 • Remove hydrophobically bound proteins, lipoproteins, and lipids by washing the column with 5–10 column volumes 30% isopropanol for about 15–20 minutes. Then wash the column with approximately 10 column volumes of distilled water.

Alternatively, wash the column with 2 column volumes of detergent in a basic or acidic solution. Use, for example, 0.1–0.5% nonionic detergent in 0.1 M acetic acid, contact time 1–2 hours. After treatment, always remove residual detergent by washing with at least 5 column volumes of 70% ethanol.

 Then wash the column with approximately 10 column volumes of distilled water. 
[[Category:Protocols]]

Ni-Beads-Regeneration - Revision history

Crm at 16:15, 7 February 2018