Editing Phosphocellulose Resin (section)

== Protocol 2 for packing PC column<br>  ==

1. Base treatment of PC: Add 50 g of dry PC to each of 5 × 2-L beakers containing 1 L of 0.5 M NaOH. Stir gently until resin is wetted and re-suspended into a uniform slurry. Let the resin settle for 5 min and then aspirate the supernatant, including any fines. Quickly add 1 L 0.5 M K-phosphate to the settled resin, stir, check that the pH is near neutral, and then let stand for 5 min. Again aspirate the supernatant, add 1 L of H2O, re-suspend the resin by stirring, and then allow to settle for 5 min prior to aspirating the supernatant. 

2. Acid treatment of PC: Add 1 L 0.5 M HCl to the settled resin, re-suspend, and allow to settle for 5 min. Aspirate the supernatant, add 1 L 0.5 M K-phosphate, stir, check the pH is near neutral, and let settle again for 5 min prior to aspirating the supernatant. Then, combine the resin in all the beakers into a single 4-L beaker and add 3 L 0.5 M K-phosphate. Allow to settle, and aspirate the supernatant. Add 1 L CB + 1 M KCl (see Item 20), stir the resin, allow to settle, and then aspirate. Repeat this wash step with 1 L CB + 1 M KCl twice more. 

3. Packing the columns: Pour the resin slurry into the column housings in the cold room and then pack the bed at ~15 mL/min using a peristaltic pump attached to the column outlet to control the flow rate. Attach a flow adapter to the top of the column housing and run 7 L of CB + 1 M KCl through at a flow rate of 5–10 mL/min. 

4. Washing, blocking, and equilibrating the column: Continue washing with 10 L of CB monitoring the conductivity to ensure that KCl is fully eluted. Adjust the level of the flow adapter to allow an adequate reserve of buffer at the top of the bed, as the resin expands when going from high to low salt. Load 300 mL of 30 mg/mL BSA in CB, chase with 700 mL of CB, and then stop the flow for 2 h to allow adsorption of the BSA. This step blocks tubulin from binding irreversibly to the fresh resin and needs to be performed only once prior to fractionating crude microtubule protein on the PC column. Finally, wash out the BSA with 2 L CB + 1 M KCl and then equilibrate the column with 10 L of CB. This last CB wash should be done in the evening before the preparation.<br> 



Regeneration of 

[[Category:Protocols]]