Table 1:
Comparison of non-radioactive hybridization methods
| Commercial protocol | Time (°C) | Homemade protocol | Time (°C) | |
|---|---|---|---|---|
| Probe hybridization | Tampon DIG Easy Hyb | Overnight (42°C) | 250 mM sodium phosphate buffer, 7% SDS, 1 mM EDTA | Overnight (65°C) |
| Hybridization washes | SSC 2×, 0.1% SDS | 2×5′ (RT) | 20 mM sodium phosphate buffer, 1% SDS, 1 mM EDTA | 2×10′ (65°C) |
| SSC 1×, 0.1% SDS | 2×15′ (60°C) | |||
| B1 washing buffer | 1′ (RT) | |||
| Membrane blocking | 10× blocking solution diluted in B2 solution | 60′ (RT) | 75 mM maleic acid pH 7.5, 200 mM NaCl, 5% non-fat dry milk powder | 60′ (RT) |
| Antibody binding | B3 10× blocking solution diluted in B2 solution | 60′ (RT) | 75 mM maleic acid pH 7.5, 200 mM NaCl, 5% non-fat dry milk powder | 30′–60′ (RT) |
| Membrane washes | B1 washing buffer | 2×15′ (RT) | 75 mM maleic acid, 200 mM NaCl, 0.3% Tween 20a | 2×15′ (RT) |
| B4 detection buffer | 5′ (RT) | 100 mM Tris pH 9.5, 100 mM NaCl | 5′ (RT) |
RT, room temperature.
a
The tween is optional, it slightly reduces the background with some probes.