Stripping and Recharging Nickel Columns
A. Set up FPLC and Strip Columns
1. Make sure there is enough filtered ddH2O, 20% EtOH, buffer A and Stripping Buffer and
that they have been recently degassed.
Stripping Buffer
20mM Sodium Phosphate
500mM NaCl
50mM EDTA pH 7.4
buffer A (loading/binding buffer):
10 mM Tris pH 8.0
5 mM MgCl2
500 mM NaCl
5 mM Imidazole
2. Remove air from inlet tubing
3. PumpWash A & B w/ ddH2O
4. Also wash out tubing w/ 1-2 ml ddH2O (in load and inject positions)
5. you can strip up to 5 columns at once by attaching them together. Make sure there is a wet
connection between each one.
Wash Column w/ ddH2O to remove ethanol (5 column volumes (CV); in load position)
6. PumpWash A & B with stripping buffer
7. Run 10 CV of stripping buffer/column @1mL/min (Will usually be stripping multiple
columns at once that will be daisy-chained together)
8. PumpWash A & B with binding buffer (A)
9. Wash Column w/ 10 CV of binding buffer (A)
10. PumpWash A & B with ddH2O
11. Wash Columns w/ 10 CV of mqH2O
12. Remove columns one at a time starting with one on the bottom to be recharged.
B. Recharge Columns and Clean FPLC
1. Recharge the water-washed column by loading 0.5 ml (for HisTrap FF 1 ml) or 2.5 ml (for a
5ml column) of 0.1 M NiSO4, respectively.
This is done by hand using sterile 10mL syringe.
2. Reattach columns to the FPLC
3. Wash columns w/ 5 CV of water
4. Move pump intakes to Buffer A; do PumpWash
5. Wash columns w/ 5 CV of buffer A (to adjust pH)
7. Move pump intakes to water; do PumpWash
8. Wash column w/ water – 5 CV
9. Move pump intakes to 20% EtOH; do PumpWash
10. Wash column w/ 20% EtOH for 5 CV; Remove column and cap and parafilm ends; store
@ 4o
11. Do final wash  PumpWash; Load Wash and Inject Wash (2-5 ml each)
12. Empty waste containers