Bio-Rad Profinity IMAC Resins - Manual (Page 45)

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1. Strip metal ions.
Wash with 10 column volumes of 50 mM sodium phosphate, 0.3 M NaCl, and
0.05–0.5 M EDTA, pH 7.5.
2. Wash the column with one of the following solutions at 2 ml/min:
• 1% acetic acid with 0.12 M phosphoric acid, pH 1.5
–
This solution may be used as a cleaning, sanitizing and storage
solution with Profinity IMAC resins
– 10–15 min exposure time
– Rinse with 10 column volumes of distilled water
• 2 M NaCl (removes ionic contaminants)
–
10–15 min exposure time
– Rinse with 10 column volumes of distilled water
• 1 M NaOH up to 3 hr (removes precipitated, hydrophobic, and
lipoproteins)
–
Exposure time is usually 1–3 hr
– Rinse with 10 column volumes of distilled water
• 70% ethanol or 30% isopropyl alcohol (removes precipitated,
hydrophobic, and lipoproteins)
–
15–20 min exposure time
– Alternatively, use 0–30% gradient isopropyl alcohol over 5 column
volumes, followed by 2 column volumes 30% isopropyl alcohol
– Rinse with 10 column volumes of distilled water
3. Remove cleaning solution(s) from column by rinsing with 10 column
volumes of binding buffer.
• Rinse column with 50 mM sodium phosphate, 0.3 M NaCl, pH 8.0
• Ensure the eluate is ~pH 8 and the UV signal has returned to the baseline
4. Recharge the column with metal ion of choice.
• Refer to Section 6, Immobilizing Metal Ions
Sanitization
Sanitization inactivates microorganisms and prevents buildup of endotoxins. For
optimal results, the column should be run at 300 cm/hr.
The column may be washed with either of the following two solutions:
• 1% acetic acid and 0.12 M phosphoric acid, pH 1.5
• 1 M NaOH
Rinse solution from column with 3–5 column volumes of distilled water.
Reequilibrate the column with 3–5 column volumes of binding buffer.
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