Editing His-MBP Fusion Protein Purification Protocol (section)

== '''Column Regeneration and Storage'''<br>  ==

=== Ni column<br>  ===

Use a peristalic pump to pump&nbsp;50~100 ml of '''Column Regeneration Buffer''' to 10 ml column: <br> 

20 mM sodium phosphate, pH 7.4, 0.5 M NaCl, 50 mM EDTA.<br> 

Wash column with 20 ml water, then pump 5~10 ml 8M Urea, wash with 100 ml water.<br> 

Charge column with 5 ml 0.1M NiSO4. <br> 

Wash with 100 ml water. 

Store column in water or 20% ethanol (long term storage) 

Before use, equilibrate column with 3CV of Buffer A. 

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The Ni2+-stripped column can be cleaned by the following methods;<br>• 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.<br> 

<br>• 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.<br> 

<br>• 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.<br> 

<br>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. <br> 

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Then wash the column with approximately 10 column volumes of distilled water.<br><br> 

For 5 ml HisTrap HP column, use half volumes. 

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=== Amylose Column<br>  ===

At room temperature or warm up the buffers to 37 oC.<br> 

<br>1. Water: 3 column volumes 

2. Wash with 3 CV of saline buffer 20 mM Tris-HCl, 200 mM NaCl and 1 mM EDTA<br>3. 0.1% SDS: 3 column volumes at room temperature<br>4. Water: 3 column volumes<br>5. PBS: 3 column volumes 

6. Store column in cold room 

Resin can be washed very briefly with 0.1 N NaOH followed by water washing step. 

Alternative method:<br>Regeneration 1: 50 mM HEPES, 4 M Urea, 0.5% w/v SDS pH 7.4.<br>Regeneration 2: 50 mM HEPES, 150 mM (NH4)2SO4, 2 mM EDTA, 2 mM EGTA pH 7.4.<br>Regenerated resin can be stored in 20% ethanol at 4 °C<br> 

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