Wiese Lab:PC Column Prep

Back to Protocols

Phosphocellulose (PC) Column Prep (300 ml)

Resin:

Whatman P11 Cellulose Phosphate -- fibrous cation exchanger (1 gram of PC swells to about 4 ml packed resin)

Summary:

To pour a 300ml column, start with 50 grams dry resin. Treat with acid/base in a 2L beaker as described below. Older procedures described the use of large Buchner funnels to rapidly remove the acid/base. However, gentle stirring of the resin with a plastic/glass rod to suspend it in a 2L beaker, followed by settling of the resin for 5 min by gravity has worked well for us. This method also incorporates defining of the resin into the acid/base cycling protocol. After acid/base treatment the resin is washed well, packed, and equilibrated for use.

Solutions & Supplies:

• 1L 0.5N NaOH
• 1L 0.5N HCl
• 4L 0.5M K-Phosphate, pH 6.8
• 5L ddH20
• 4L CB + 1M KCl
• 10L CB
• 50 grams Phosphocellulose
• 4L beaker
• 1-1.5L cleaned column housing
• Stirring rod
• Peristaltic pump
• Aspirators with large trap

Column Preparation Procedure:

1. Pour 1L 0.5N NaOH into 4L beakers. Add 50 grams PC to the beaker stirring gently with a rod until the PC is wetted and an even slurry is present. Let stand at room temperature for 5 min.
2. Aspirate off supernatant, including fines, and quickly add 1L 0.5M K-phosphate to neutralize, gently mixing with a rod. Check that pH is ~7 and let stand 5 min.
3. Aspirate off supernatant, add 1L ddH20 and gently stir to resuspend settled resin. Allow the resin to settle.
4. Aspirate off supernatant, add 1L 0.5N HCl, gently stir to resuspend and wait 5 min.
5. Aspirate off supernatant, add 1L 0.5M K-phosphate, stir and check pH is 7.
6. After resin has settled, aspirate supernatant. Use the remaining 0.5M K-phosphate to wash the resin by resuspending, letting settle and aspirating the supernatant.
7. Wash 3 x 1L CB + 1M KCl as done in 7.
8. In the cold room, pour the resuspended resin into the column housing (with a mark approx. 50 cm high in a 5 cm ID housing) and pack by pumping from the bottom (i.e. the peristaltic pump is "sucking" buffer from the bottom of the column and depositing it into a waste jug). Pack at 45 ml/hour/cm cross-sectional area. For a 5 cm diameter column this is ~880 ml/hour or ~14.5 ml/min. After resin is packed, switch to pumping from the top. Run 1L of CB + 1M KCl through the column at 5-10 ml/min.
9. Wash with 2L CB. Check conductivity to ensure that all the KCl is gone. The resin may expand as the salt is washed out so make sure there is a large buffer head on the resin bed. The column is now ready for use.