Stripping and Recharging Nickel Columns

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 à PumpWash; Load Wash and Inject Wash (2-5 ml each)

12. Empty waste containers