Western Blot Protocol:1. SDS electrophoresis2. Transfer (use Immobilon PVDF or Hybond nitro-cellulose (Amersham) - activate in methanol for 1-2 minutes - wash with water for 5 minutes - incubate in transfer buffer for 5 minutes (15,1g TRIS, 72,1g glycine, 1000ml methanol, add water up to final 5000ml, pH8,5) - bring gel/membrane/3MM paper together - transfer over night (330 mA for 28x18 cm gels) or equivalent3. Immunoblot - block with PBS/10% non-fat dry milk/0,3% Tween 20 over night at 4°C or 1 hour at 37°C - wash 2x with PBS/0,3% Tween 20 for 5 minutes - 10µg/ml in PBS/5% non-fat dry milk/0,3% Tween 20-> If you do not see anything at this concentration there is not enough detectable protein, under normal conditions 1µg/ml may be sufficient. - incubate 1 hour at room temperature - wash 4x with PBS/0,3% Tween 20 for 5 minutes- second antibody: rabbit anti-mouse-HRPO (DAKO) 1:1’000 in PBS/5% non-fat dry milk/0,3% Tween 20 for 1hour at room temperature - wash 4x with PBS/0,3% Tween 20 for 5 minutes4. detection with ECL system from Amersham - mix sol1 and sol2 in equal amounts - incubate blot for 1 minute at room temperature in this mixture - dry blot carefully with tissue - cover blot in Saran Wrap - lay "side of gel" to X-ray film, expose for 5 seconds to 10 minutes.
