Editing Phosphocellulose Resin (section)

== Pouring a 1L Phosphocellulose (PC) Column Resin<br>  ==

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

Summary: To pour a 1L column, start with 220 grams dry resin divided into 5 aliquots of 44 grams. Treat each aliquot 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' by gravity has worked well for us. This method also incorporates de-fining of the resin into the acid/base cycling protocol. After acid/base treatment the resin is washed well, packed, treated with BSA to block irreversible binding sites and equilibrated for use. 

Solutions &amp; Supplies: 220 grams Phosphocellulose 5L 0.5N NaOH 5L 0.5N HCl 13L 0.5M K-Phosphate, pH 6.8 5L ddH20 5 2L beakers 12L CB + 1M KCl 20L CB 300 ml of 30 mg/ml BSA in CB (filtered) 1-1.5L cleaned column housing 2 stirring rods 2 aspirators with large traps Peristaltic pump 10 ml plastic pipets as inlets for peristaltic pump 

Column Preparation Procedure: 

1. Pour 1L 0.5N NaOH into 5 x 2L beakers. Add 44 grams PC to each 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. 

4. Allow the resin to settle. 

5. Aspirate off supernatant, add 1L 0.5N HCl, gently stir to resuspend and wait 5 min. 

6. Aspirate off supernatant, add 1L 0.5M K-phosphate, stir and check pH is 7. 

7. After resin has settled, aspirate supernatant and combine all the resin in a 4L beaker. Use the remaining 0.5M K-phosphate to wash the resin by resuspending, letting settle and aspirating the supernatant. 

8. Wash 3 x 1L CB + 1M KCl as done in 7. 

9. 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 7L of CB + 1M KCl through the column at 5-10 ml/min. 

10. Wash with 10L 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. 

11. Load 300 ml of 30 mg/ml BSA (Fraction V; filtered) in CB, follow with 700 ml CB and stop the column. Leave the column sitting for 2 hr during which the BSA blocks irreversible binding sites on the resin -- this is very important the first time a column is used to prevent loss of the tubulin. 

12. Wash the column with 2L CB + 1M KCl to elute BSA that is not irreversibly bound. 

13. Wash column with 10L of CB. The column is now ready for use. 

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