Phosphocellulose Resin

Purification of Restriction Enzymes on Phosphocellulose Column

AluI, BamI, BglI, BglII, EcoRI, EcoRII, HaeII, and HaeIII, HgaI, HincII, HsuI (isoschizomer of HindIII), PstI, SalI, TaqI, HpaI and HpaII.

Preparation PC

1. Suspend 125 g P11 PC in 4 L 0f 0.2 N HCl diluted with (1:1) 95% EtOH, stir gently for 30 min at RT.
2. Settle and aspirate/decant supernatant to remove fines.
3. Collect PC by filtration.
4. Wash 3 ~ 4 times with 4 L distill water.
5. Adjust pH to neutral with 1 M NaOH
6. Suspend PC in 4 L 0.1 N NaOH
7. Stir for 30 min at RT.
8. Collect PC by filtration.
9. Suspend in 4 L of 1 mM EDTA.
10. Stir for 30 min at RT.
11. Wash with 4 L water.
12. Adjust pH to neutral with 1 M HCl.
13. Resuspend in Buffer PC containing 0.2 M NaCl.
14. Adjust pH to 7.0.
15. Pour to column--> 500 ml packed column bed volume
16. Equilibrate column with several column volume of Buffer PC containing 0.2 M NaCl

Purification of Restriction Enzymes

1. Lyse 50 g cells in 200 ml of 10 mM K2HPO4-KH2P04 pH 7.0, 5 mM mercaptoethanol, 1 mM EDTA, O.2M NaCl.
2. Spin and load cleared cell lysate supernatant to PC column (with 5 ml guard column):
150~200 ml bed volume (2.5 X 35 cm) is enough for 50 g cells.
3. Wash with several CV of Buffer PC until A280 -> ~ 0.
4. Elute with 7CV of linear gradient (1L ~ 1.5 L) from 0.2 to 1 M NaCl.
5. Collect 10 ml/fraction.
6. Combine fractions and load to Ni column.
7. Wash with Buffer A 35 mM Imidazole
8. Elute with Buffer B containing 300 mM Imidazole

9. Collect fractions, anaylse fraction for enzyme activity,

10. Concentrate fractions containing active enzyme using spin concentrator. Analyse enzyme purity by SDS-PAGE

11. Dialyse against enzyme storage buffer containing 50% glycerol.

12. Store enzyme at -20 oC or -80 oC.

If enzyme is not pure enough, do purification on Q HP, SP HP, HYDROXYLAPATITE or Biorex 70:

For example:

• Dialyse enzymes with  Buffer PC (10 mM K2HPO4-KH2P04 pH 7.0, 5 mM mercaptoethanol, 1 mM EDTA, O.2M NaCl), or buffer exchange using Sephadex G25.
  
• Load to 20 ml hydroxylapatite column
  
• Wash with 100 ml Buffer PC (10 mM K2HPO4-KH2P04 pH 7.0, 5 mM mercaptoethanol, 1 mM EDTA, O.2M NaCl).
  
• Elute with 300 ml gradient of 10 mM potassium phosphate pH 7.0 containing 1 mM EDTA, 7mM b-ME, from 0.2M to 1 M NaCl
  
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Biorex 70:

Enzyme could be in flow through or required 0 to 0.5 M NaCl gradient elution

Pouring a 1L Phosphocellulose (PC) Column Resin

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 & 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.

Protocol 2 for packing PC column

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.

Regeneration of