14. TEMED.
15. Ethanol.
16. Blotting paper.
17. SDS-PAGE gel-running tank.
18. 10Â Tris-glycine buffer: 250 mM Tris base, 1.92 M glycine.
19. SDS solution.
20. Migration buffer: 1Â Tris-glycine, 0.1% SDS.
21. SDS-PAGE protein size ruler/marker.
22. Power generator.
23. PVDF membrane, gel sized.
24. 80% Ethanol.
25. Blotting paper, gel sized.
26. SDS-PAGE electro-transfer system.
27. Transfer buffer: 1Â Tris-glycine, 20% ethanol.
28. Magnetic stir bar.
29. Magnetic stirrer at 4
C.
30. Crystal box large enough to fit PVDF membrane.
31. 1Â PBS (see Subheading 2.2).
32. PBS/Tween: 1Â PBS, 0.1% Tween.
33. Benchtop oscillator.
34. PBS/Tween/milk: 1Â PBS, 0.1% Tween, 5% powdered milk.
35. Anti-GFP antibody (as in ref. 8, or commercially available
antibodies).
36. Appropriate secondary antibody coupled to horseradish
peroxidase.
37. Transparent plastic envelope/pouch.
38. ECL chemiluminescence substrate.
39. Equipment to detect chemiluminescence (film or digital
appliance).
40. Coomassie Brilliant Blue R-250.
41. Glacial acetic acid.
42. Coomassie staining buffer: 40% Ethanol, 10% glacial acetic
acid, 0.1% Coomassie Brilliant Blue R-250.
43. Destaining buffer: 40% Ethanol, 10% glacial acetic acid.
2.6 Subcellular
Localization
of dsRNA-associated
Protein Candidates
1. N. benthamiana plants expressing the 35S:B2:GFP cassette
[7]: These plants are available upon request. Plants should be
3–4 weeks old and not watered for 1–2 days prior to
infiltration.
312
Marco Incarbone and Christophe Ritzenthaler
15. Ethanol.
16. Blotting paper.
17. SDS-PAGE gel-running tank.
18. 10Â Tris-glycine buffer: 250 mM Tris base, 1.92 M glycine.
19. SDS solution.
20. Migration buffer: 1Â Tris-glycine, 0.1% SDS.
21. SDS-PAGE protein size ruler/marker.
22. Power generator.
23. PVDF membrane, gel sized.
24. 80% Ethanol.
25. Blotting paper, gel sized.
26. SDS-PAGE electro-transfer system.
27. Transfer buffer: 1Â Tris-glycine, 20% ethanol.
28. Magnetic stir bar.
29. Magnetic stirrer at 4
C.
30. Crystal box large enough to fit PVDF membrane.
31. 1Â PBS (see Subheading 2.2).
32. PBS/Tween: 1Â PBS, 0.1% Tween.
33. Benchtop oscillator.
34. PBS/Tween/milk: 1Â PBS, 0.1% Tween, 5% powdered milk.
35. Anti-GFP antibody (as in ref. 8, or commercially available
antibodies).
36. Appropriate secondary antibody coupled to horseradish
peroxidase.
37. Transparent plastic envelope/pouch.
38. ECL chemiluminescence substrate.
39. Equipment to detect chemiluminescence (film or digital
appliance).
40. Coomassie Brilliant Blue R-250.
41. Glacial acetic acid.
42. Coomassie staining buffer: 40% Ethanol, 10% glacial acetic
acid, 0.1% Coomassie Brilliant Blue R-250.
43. Destaining buffer: 40% Ethanol, 10% glacial acetic acid.
2.6 Subcellular
Localization
of dsRNA-associated
Protein Candidates
1. N. benthamiana plants expressing the 35S:B2:GFP cassette
[7]: These plants are available upon request. Plants should be
3–4 weeks old and not watered for 1–2 days prior to
infiltration.
312
Marco Incarbone and Christophe Ritzenthaler