Purification of Starch Granules from Arabidopsis Leaves and Determination of Granule-Bound Starch Synthase Activity

Materials and Reagents

1. Plant materials
   Note: Arabidopsis thaliana (A. thaliana) were grown in controlled cabinets on peat-based compost.
   
2. Liquid N₂
   
3. HEPES (Sigma-Aldrich, catalog number: H4034 )
   
4. Potassium hydroxide pellets (Panreac Applichem, catalog number: A0566 )
   
5. Triton^TM X-100 (Sigma-Aldrich, catalog number: X100 )
   
6. Miracloth (Merck KGaA, catalog number: 475855 )
   
7. Percoll^® (Sigma-Aldrich, catalog number: P1644 )
   
8. Glycogen (from rabbit liver Type III) (Sigma-Aldrich, catalog number: G8876 )
   
9. Tricine (Sigma-Aldrich, catalog number: T0377 )
   
10. Potassium acetate (Sigma-Aldrich, catalog number: P1147 )
    
11. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
    
12. Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Sigma-Aldrich, catalog number: E5134 )
    
13. Sodium hydroxide pellets (Panreac Applichem, catalog number: 131687 )
    
14. Maltotriose (Sigma-Aldrich, catalog number: M8378 )
    
15. Adenosine 5’ diphospho, [D-glucose-14C(U)] ammonium salt (ADP [U-14C] glucose) (American Radiolabeled Chemicals, catalog number: ARC 3297 )
    
16. Methanol (CARLO ERBA Reagents, catalog number: 412532 )
    
17. Potassium chloride (AppliChem GmbH, catalog number: A2939 )
    
18. Ecolite(+)^TM liquid scintillation cocktail (MP Biomedicals, catalog number: 882475 )
    
19. 1 M HEPES-KOH (see Recipes)
    
20. 1 M Tricine (see Recipes)
    
21. 0.25 M Potassium acetate (see Recipes)
    
22. 1 M DTT (see Recipes)
    
23. 0.5 M EDTA (see Recipes)
    
24. 0.1 M Maltotriose (see Recipes)
    
25. 3 M KCl (see Recipes)
    
26. 20 mg/ml Glycogen solution (see Recipes)
    
27. Extraction buffer (see Recipes)
    
28. Percoll buffer (see Recipes)
    
29. Washing buffer (see Recipes)
    
30. Precipitation buffer (see Recipes)
    

Equipment

1. Small mortar and pestle
   
2. 1.5 ml microfuge tubes
   
3. Automatic pipettes
   
4. Precision scale
   
5. Centrifuge (Eppendorf, model: 5810 R )
   
6. Microcentrifuge (Eppendorf, model: 5424 )
   
7. Scintillation Counter (Beckman Coulter, model: LS 6000 IC )
   
8. Scintillation vials (Sigma-Aldrich, catalog number: Z376817 )
   

Procedure