In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves

Materials and Reagents

1. Commonly-used medical cotton gauze
   
2. 0.2 ml tube
   
3. Vallisneria plants
   Note: Young plants of Vallisneria of 20-30 cm long were purchased at a tropical-fish store and cultured to be grown over 100 cm in buckets (20 L) filled with tap water and with a layer of soil at the bottom (Figure 1A). The plants were grown under 12 h light/12 h dark regime at 20-24 °C.
   
4. Dextran (from Leuconostoc ssp., Mr ~500,000) (Fluka, catalog number: 31392 )
   Note: Currently, it is “Sigma-Aldrich, catalog number: 31392 ”.
   
5. Polyethylene glycol (Sigma-Aldrich, catalog number: P-3640 )
   
6. SDS-PAGE
   
7. Deionized water
   
8. 1% Triton X-100 (Sigma-Aldrich, catalog number: T-9284 )
   
9. 50 mM MOPS-KOH (pH7.6) (DOJINDO, catalog number: 343-01805 )
   
10. 300 mM sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 196-00015 )
    
11. 10 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N’,N’,-tetraacetic acid (EGTA) (DOJINDO, catalog number: 342-01314 )
    
12. 5 mM ethylenediaminetetraacetic acid (EDTA) (DOJINDO, catalog number: 345-01865 )
    
13. 10 μg/ml dibutylhydroxytoluene (Tokyo Chemical Industry, catalog number: D0228 )
    
14. 1% casein (Nacalai tesque, catalog number: 073-19 )
    
15. 5 mM K₂S₂O₅ (Wako Pure Chemical Industries, Siyaku, catalog number: 161-03345 )
    
16. 1 mM dithiothreitol (DTT) (Wako Pure Chemical Industries, catalog number: 048-29224 )
    
17. 2.5 mg/ml pepstatin A (Sigma-Aldrich, catalog number: P-4265 )
    
18. 2.5 mg/ml aprotinin (Sigma-Aldrich, catalog number: A-4529 )
    
19. 20 mg/ml EDTA-washed Polyvinylpolypyrolidone (Sigma-Aldrich, catalog number: P-6755 )
    
20. MOPS-KOH (DOJINDO, catalog number: 345-02225 )
    
21. 25 mM MgCl₂ (Wako Pure Chemical Industries, Siyaku, catalog number: 135-00165 )
    
22. 250 mM KCl (Wako Pure Chemical Industries, Siyaku, catalog number: 163-03545 )
    
23. 40% glycerol (Wako Pure Chemical Industries, Siyaku, catalog number: 075-00616 )
    
24. 20% 2-mercaptoethanol (Sigma-Aldrich, catalog number: A-6365 )
    
25. A small amount of bromophenol blue (Wako Pure Chemical Industries, Siyaku, catalog number: 021-02911 )
    
26. 0.5 M Tris-HCl (pH 6.8) (Sigma-Aldrich, catalog number: 252859 )
    
27. Homogenizing medium (see Recipes)
    
28. Buffer A (see Recipes)
    
29. Buffer B (see Recipes)
    
30. 5x phosphorylation buffer (see Recipes)
    
31. ATP mixture (see Recipes)
    
32. 4x SDS buffer (see Recipes)
    

Equipment

1. Blade (Lion Office Products Corp., model: L-300 )
   
2. Polytron homogenizer (Kinematica AG, model: PT-35/ 2ST”OD” )
   
3. Centrifuge
   
4. Ultracentrifuge
   
5. Teflon homogenizer (10 ml) (Ikemoto Scientific Technology, model: 812-771-04 )
   
6. Lighting system (Sugiura Lab, model: FI-150T )
   
7. Cut-off filter (KenkoTokina Corporation, model: Y-44 )
   
8. Interference filter (KenkoTokina Corporation, model: BP-45 )
   
9. Autoradiography (Fujifilm Corporation)
   

Procedure