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. Blotting papers in transfer buffer 10 min
• Sandwich (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!