Editing Prepare-Cel1-from-Celery (section)

== '''Protocol 3'''<br>  ==

1. Use a Blender to chop Celery stalks (7 kg) at 4 <sup>o</sup>C.<br>2. Adjusted with a 10Ã solution to give the composition of buffer A (0.1 M TrisâHCl, pH 7.7, 10 mM phenylmethanesulfonyl<br>fluoride (PMSF).<br>3. Spin for 20 min at 6000 rpm to pellet debris.<br>4. Add (NH4)2SO4 to supernatant to adjust to 20% saturation.<br>5. Put on ice for 30 min.<br>6. Spin at 12000 rpm for 30 min at 4°C<br>7. Add (NH4)2SO4 to the resulting supernatant to 70% saturation.<br>8. Put on ice for 30 min.<br>9. Spin at 25000 rpm for 30 min. 

10. Dissolve the final pellet in 250 ml buffer A and dialyze against 0.5 M KCl in buffer A. 

11. Incubate the solution with 10 ml concanavalin AâSepharose resin (Sigma) overnight at 4 oC. 

12. Pack the slurry into a 2.5 cm diameter column and wash with 0.5 M KCl in buffer A. 

13. Elute bound CEL-I with 90 ml 0.3 M a-D+-mannose, 0.5 M KCl in buffer A at 65 oC. 

14. Dialyse CEL-I against buffer B (25 mM potassium phosphate, 10 mM PMSF, pH 7.0). 

15. Apply the dialysed solution to a 100 ml phosphocellulose P-11 column that had been equilibrated in buffer B. 

16. Elute the bound enzyme with a linear gradient of KCl in buffer B. 

17. Pool fractions containing&nbsp; CEL-I activity. 

18. Add ammonium sulfate to saturation, incubate on ice for 30 min. 

19. Spin to collect precipitate. 

20. Dissolve the precipitate in Buffer C (50 mM TrisâHCl, pH 7.8, 0.2 M KCl, 10 mM PMSF, 1 mM ZnCl<sub>2</sub>). 

21. Dialyse against Buffer C. 

22. Load to a Superose 12 FPLC column equilibrated with Buffer C.<br>