Western blotting protocol

Solutions

• Transfer buffer (“Towbin”): 1L: 3g Tris, 14.4g glycine, 800ml H2O, 200ml MeOH
• TBS: 5X: 100ml 1M Tris-HCl pH 7.5, 150ml 5M NaCl, 750ml ddH2O. Sterile filter after diluting to 1x. Store at 4°C.
• TBST: 1L TBS, 500ul of Tween-20 (0.05%)
• Blocking buffer: 1.5g BSA (3%), 50ml TBST (alternative: 5% nonfat milk powder)

Protocol

• Cut away stacking gel, cut corner for orientation, and soak in transfer buffer 45 min.
• Cut PVDF and four 3mm Whatman blotting paper to correct size. Cut corner of PVDF.
• Wet PVDF with methanol, soak 5 minutes in H20, then 10 minutes in transfer buffer. Leave the blotting papers in transfer buffer for 10 min, shaking gently.
• Transfer: Sandwich the gel and membrane between the blotting papers and start the power (protein moves towards (+) anode... not the same each instrument). Note orientation.
• Block: 1 hr on rocker with 5% milk/TBST. Use the lid of tip box and 15 mL fluid to cover blot.
• Wash: 10 minutes 3X in 15 mL TBST.
• Secondary antibody: incubate 30 minutes with antibody in TBST (0.1%). 10-50 ng/ml.
• Wash: 10 minutes 3X in 15 mL TBST.
• Image!

Important Note The electrodes on the trays and inside of the turboblot system are sensitive to salt buildup from the buffers. After using, use a wet kimwipe to remove all buffer inside the tray. Also wipe down the electrodes on the lid, tray, and inside of the system (that the trays connect to) to prevent salt buildup