2.3
North-Western Blot
1. 10Â HEPES buffer: 200 mM HEPES, 10 mM EDTA. Adjust
to pH 7.8 with 1 M KOH, and sterilize by filtration.
2. Agarose (molecular biology grade).
3. 4Â RNA-loading buffer: 50% Glycerol, 50 mM Tris–HCl
pH 7.5, 5 mM EDTA, bromophenol blue, 50 μg/mL ethidium bromide.
4. Gel-running tank at 4
C.
5. Power generator.
6. Gel documentation and analysis equipment (e.g.: Gel Doc).
7. Capillary transfer apparatus.
8. 20Â SSC: 3 M NaCl, 0.3 M sodium citrate. Sterilize by
filtration.
9. Blotting paper.
10. Nylon membrane, gel sized (e.g.: Amersham HyBond™ N+).
11. Stack of paper towels.
12. Approximately 1 kg weight.
13. 2Â SSC.
14. UV cross-linker.
15. Crystal box large enough to accommodate the nylon
membrane.
16. 1Â PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ,
1.8 mM KH 2 PO 4 . Adjust to pH 7.4 with HCl.
17. PBS/Tween: 1Â PBS, 0.1% Tween.
18. PBS/Tween/milk: 1Â PBS, 0.1% Tween, 5% powdered milk.
19. Benchtop oscillator.
20. Recombinant B2:StrepTagII [7]: The plasmid to produce this
protein is available upon request.
21. StrepTactin conjugated to horseradish peroxidase (IBA Life
Sciences Strep-Tactin
® -HRP conjugate).
22. Transparent plastic folder.
23. ECL chemiluminescence substrate.
24. Equipment to detect chemiluminescence (film or digital
appliance).
2.4 Northern Blot
1. Subheadings 2.3, items 1–3 and 5–15.
2. Gel electrophoresis tank.
3. Formaldehyde.
4. RNA denaturing loading buffer: 1Â HEPES buffer, 50% formamide, 6% formaldehyde, 20% glycerol, 0.05% (w/v) bromophenol blue, 50 μg/mL ethidium bromide.
310
Marco Incarbone and Christophe Ritzenthaler
North-Western Blot
1. 10Â HEPES buffer: 200 mM HEPES, 10 mM EDTA. Adjust
to pH 7.8 with 1 M KOH, and sterilize by filtration.
2. Agarose (molecular biology grade).
3. 4Â RNA-loading buffer: 50% Glycerol, 50 mM Tris–HCl
pH 7.5, 5 mM EDTA, bromophenol blue, 50 μg/mL ethidium bromide.
4. Gel-running tank at 4
C.
5. Power generator.
6. Gel documentation and analysis equipment (e.g.: Gel Doc).
7. Capillary transfer apparatus.
8. 20Â SSC: 3 M NaCl, 0.3 M sodium citrate. Sterilize by
filtration.
9. Blotting paper.
10. Nylon membrane, gel sized (e.g.: Amersham HyBond™ N+).
11. Stack of paper towels.
12. Approximately 1 kg weight.
13. 2Â SSC.
14. UV cross-linker.
15. Crystal box large enough to accommodate the nylon
membrane.
16. 1Â PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ,
1.8 mM KH 2 PO 4 . Adjust to pH 7.4 with HCl.
17. PBS/Tween: 1Â PBS, 0.1% Tween.
18. PBS/Tween/milk: 1Â PBS, 0.1% Tween, 5% powdered milk.
19. Benchtop oscillator.
20. Recombinant B2:StrepTagII [7]: The plasmid to produce this
protein is available upon request.
21. StrepTactin conjugated to horseradish peroxidase (IBA Life
Sciences Strep-Tactin
® -HRP conjugate).
22. Transparent plastic folder.
23. ECL chemiluminescence substrate.
24. Equipment to detect chemiluminescence (film or digital
appliance).
2.4 Northern Blot
1. Subheadings 2.3, items 1–3 and 5–15.
2. Gel electrophoresis tank.
3. Formaldehyde.
4. RNA denaturing loading buffer: 1Â HEPES buffer, 50% formamide, 6% formaldehyde, 20% glycerol, 0.05% (w/v) bromophenol blue, 50 μg/mL ethidium bromide.
310
Marco Incarbone and Christophe Ritzenthaler