- Sample preparation
Lysis of sample in appropriate lysis buffer (eg RIPA or directly Leammli)
- Reduce and denature sample
Add sample buffer (SDS and β-mercaptoethanol). Heat 95 ̊C 5 min (sonicate a few sec to break DNA)
- Loading/running the gel
- 30 – 50 μg whole lysate per laneor 100 ng purified protein
Optimize amount depending on expression level of the protein
- Run the gel (mini gel 35 mA)
(25 mM Tris, 200 mM Glycine, 1% SDS)
- Transfer proteins from the gel to membrane
Used nitrocellulose (500 mA 1h-1.30h, wet system)
(20 mM Tris, 150 mM Glycine, 0.1% SDS, 10% MeOH)
- Blocking
Incubate membrane in 2% BSA or OVA for 1 h (25 ml for small gel) (diluted in PBS 0.05% Tween-20)
- Primary antibody incubation
- Incubate membrane in primary antibody (at recommended concentration and diluted in PBS 0.05% Tween-20) 1-2 hr RT or preferentially overnight at 4 ̊C
- Test 2-3 dilutions (1/1,000-1/5,000; 1-10 µg/ml) (inc in 5 ml PBS-Tween-20 using 50 ml Falcon tube) (shaking/rolling)
- Wash 3x PBS 0.05% Tween20, 5 min-10 min each (shaking)
- Secondary antibody incubation
- Incubate with enzyme (eg HRP) conjugated secondary Antibody (diluted in PBS 0.05%Tween-20 1% BSA or OVA) 1 hr RTat 1/10,000) (Biorad anti-goat, 0.5 µl/mini-gel, 15-20 ml/gel) (shaking/rolling)
- Wash 3x PBS 0.05% Tween-20, 5 min-10 min each (shaking)
- ECL detection