Immunoblotting

Detection of lung IgA by western-blotting

Material

• 100kD Amicon® Ultra centrifugal filters; Merck Millipore
• Laemmli sample buffer (βmercapto included 5% in reducing conditions) 4X
• Migration buffer : TrisGlycine 10X, diluted in water (keep it after use, Bio-Rad)
• Mini-protean pre-cast gels 4-15% polyacrylamide, Bio-Rad
• Protein standard : PageRuler Prestained Protein Ladder 26616 ThermoScientific
• Clarity^TM Western ECL, Bio-Rad

Protein migration

• Concentrate BAL samples with Amicon® Ultra centrifugal filters (adjust time and spin speed according to BAL volume and viscosity)
• Add Laemmli sample buffer 4X to ≈ 1ug  of protein and heat 5min at 95°C
• After heating, store samples in ice while loading 25ul of samples in dedicated well (see table below)
• Migration : 60 minutes, 120V

Transfer

• Equilibrate nitrocellulose membrane in transfer buffer
• Rinse gel briefly in water and equilibrate gel in transfer medium for 15 minutes
• Place one wet filter paper on the semi-dry transfer apparatus (anode side)
• Place membrane on the top of the paper, then gel on the membrane.
• Place the second piece of filter paper on the top of the gel, roll out any bubbles
• Place the cathode assembly onto the transfer stack and the safety cover.
• Transfer proteins for 20 min, 22V

Protein detection

• Rinse membrane in ultrapure water
• Wet in PBS 1X for 2 minutes
• Block the membrane in PBS 1X + 5% non fat milk overnight, 4°C
• Wash in PBS 1X-0.1% Tween for 5 minutes (stirring is needed). Repeat two times
• Add secondary antibody diluted in PBS 1X + 5% non fat milk (horseradish peroxidase-conjugated goat anti-human IgA used at a 1:20,000) for 1 hour at room temperature
• Wash in PBS 1X -0.1% Tween for 5 minutes (stirring is needed). Repeat two times
• Wash in PBS 1X for 5 minutes
• Add 5ml of Enhanced Chemi-Luminescence (ECL) substrate (prepared immediately before use) for 5 minutes
• Read membrane with a camera system, i.e.: ImageQuant^TM LAS4000, GE Healthcare

Category