Wiese Lab:PC Column Prep
Phosphocellulose (PC) Column Prep (300 ml)
Whatman P11 Cellulose Phosphate -- fibrous cation exchanger (1 gram of PC swells to about 4 ml packed resin)
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:
- 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.
- 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.
- Aspirate off supernatant, add 1L ddH20 and gently stir to resuspend settled resin. Allow the resin to settle.
- Aspirate off supernatant, add 1L 0.5N HCl, gently stir to resuspend and wait 5 min.
- Aspirate off supernatant, add 1L 0.5M K-phosphate, stir and check pH is 7.
- 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.
- Wash 3 x 1L CB + 1M KCl as done in 7.
- 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.
- 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.