Protein Degradation Assays in Arabidopsis Protoplasts

Materials and Reagents

1. 3-week old Arabidopsis plants
   
2. Mannitol (BDH Prolabo, catalog number: 25311 )
   
3. CaCl₂ (POCH, catalog number: M00015143 )
   
4. KCl (USB, catalog number: 20598 )
   
5. 2-(N-morpholino)ethanesulfonic acid (MES) (LabEmpire, catalog number: MES503 )
   
6. NaCl (POCH, catalog number: BA4121116 )
   
7. Polyethylene glycol (PEG) 4000 (Sigma-Aldrich, catalog number: 81240 )
   
8. Cellulase (SERVA Electrophoresis GmbH, catalog number: 16419 )
   
9. Macerozyme (SERVA Electrophoresis GmbH, catalog number: 28302 )
   
10. GenElute^TM HP Plasmid Midiprep Kit (Sigma-Aldrich, catalog number: PLD35 )
    
11. Benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal, Z-Leu-Leu-Leu-al (MG132) (Sigma-Aldrich, catalog number: M7449 )
    
12. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
    
13. HEPES buffer (Sigma-Aldrich, catalog number: H3375 )
    
14. MgCl₂ (USB, catalog number: 18641 )
    
15. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
    
16. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
    
17. Protease inhibitors (Roche Diagnostics, catalog number: 11873580001 )
    
18. Enzyme solution (see Recipes)
    
19. W5 (see Recipes)
    
20. W1 (see Recipes)
    
21. MMg (see Recipes)
    
22. 40% PEG (see Recipes)
    
23. Protein isolation buffer (see Recipes)
    

Equipment

1. Light microscope (Nikon Corporation)
   
2. Hemocythometer (Sigma-Aldrich)
   
3. Tape (Scotch^® Magic^TM)
   
4. Stainless steel forceps (Sigma-Aldrich)
   
5. Scissors
   
6. 10 ml pipette
   
7. 50 ml tubes
   
8. 90 mm Petri dishes
   
9. Amicon Ultra-15 centrifugal filter unit (Millipore)
   
10. NanoDrop spectrophotometer
    
11. Tabletop centrifuge
    
12. Horizontal shaker
    

Procedure