Western Blot using Rapid Histone SDS-PAGE extraction buffer
- Freshly prepare extraction buffer:
| final conc. | for 10 ml | for 5 ml |
| 1M TRIS/Cl ph6.8 | 62.5 mM | 0.625 ml | 0.312 ml |
| 20% SDS | 3% | 1.5 ml | 0.75 ml |
| sucrose | 10% | 1 g | 0.5 g |
| DTT (154 g/mol) | 0.2 M | 0.3 g | 0.15 g |
| 2.5M Na-butyrate | 1.25 mM | 5 µl | 2.5 µl |
- add tissue sample into 200-500 µl extraction buffer
- sonicate with 70% amplitude, 3 times 15 seconds, on ice
- cool on ice for 30 seconds between each sonication step
- 10 µl sample + 5 µl 3x Laemmli-buffer
- denature at 99°C for 5 minutes
- SDS-PAGE 15% (20 mA for stacking, 40 mA for separation)
- Transfer by Transblot (7 min)
- block membrane in TTBS + 5% fat-free dry milk, 1 h at RT
- proceed to Western Blot
Stripping solution
| final conc. | for 50 ml |
| 1M TRIS/Cl ph6.8 | 62 mM | 3.1 ml |
| 20% SDS | 2% | 5 ml |
| Beta-Mercaptoethanol | 0.8 % | 0.4 ml |
- incubate at 50°C for 1 hour in a tightly closed 50 ml Falcon tube
- wash with destilled water until smell of Beta-Mercaptoethanol has completely disappeared (Beta Mercaptoethanol (disulfide-reducing agent) is bad for antibodies)
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