SDS-PAGE and Immunoblotting Tim Corson, PhD Indiana University School of Medicine http://iueye.iu.edu/corson November 2010 SDS-PAGE 1.

Prepare bis-tris gels or use precast TGX gels (Bio-Rad) 2. Dilute samples appropriately in NuPAGE sample buffer (for bis-tris) or Laemmli buffer (for TGX) 3. Boil samples 5 minutes at 100C in heating block 4. Load 10-50 L protein sample per well 5. Add appropriate running buffer to tank 6. Run gel 200 V, 0.5 hour 7. Remove gel from glass or plastic plates, cut off stacking gel Transfer 1. Soak 69 cm nitrocellulose membrane in transfer buffer appropriate for gel system (use forceps!) 2. Prepare transfer sandwich: soak sponges in buffer, layer a buffer-soaked blotting paper sheet (710 cm) on top, roll out bubbles with a large test tube 3. Layer gel on top of paper, roll out bubbles 4. Carefully place membrane on top of gel 5. Layer another soaked blotting paper square on top, roll out bubbles 6. Add sponge 7. Close sandwich, place in transfer unit, with membrane side closest to positive electrode (red) 8. Transfer 75 V, 90 min Staining 1. After transfer, gels and blots may be stained 2. Shake gel in 5-10 mL Coomassie Blue (recyclable!) for 10 minutes, followed by two 15 minute washes with water to destain, leaving dark blue protein bands 3. To stain membrane: Shake in 5 mL Ponceau S (recyclable!) for 5 minutes, 4. Rinse thoroughly with water to visualize pink protein bands 5. Cut or mark gel bands as necessary now; stain will disappear during blocking Immunoblotting 1. Block membrane by shaking in 5 mL 5% skim milk powder in TBST, 1 hour 2. Replace blocking solution with a similar solution containing an appropriate dilution of primary antibody 3. Shake for 1-3 hours at RT OR shake at 4C overnight (covered) 4. Remove and save antibody solution (preserve with 0.02% sodium azide) 5. Rinse blot twice with TBST 6. Wash three times for five minutes in TBST 7. Prepare secondary antibody dilution in 5% skim milk powder in TBST
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8. Add secondary antibody solution, incubate with shaking 1 hour at RT 9. Discard antibody solution, and rinse blot twice with TBST 10. Wash five times for five minutes in TBST 11. Cover with ECL+ reagent (25 L Solution B + 975 L Solution A per blot) 12. Transfer to GelDoc or film cassette (in Saran wrap) for imaging RECIPES 2 Laemmli sample buffer 1514.2 mg Tris pH 6.8 (final 1 = 62.5 mM) 6 g SDS in 50 mL H2O (final = 3%) 40 mL glycerol (final = 20%) 10 mL b-mercaptoethanol (final = 5%) 4 Running buffer for TGX Gels For 2 L: 24g Tris base (final 1 = 25 mM) 115.2 g Glycine (final = 192 mM) pH should be 8.3. For each gel tank, make 400 mL of 1 in H2O, add 4 mL (0.1%) 10% SDS Towbin Transfer buffer for TGX Gels For 6 L: 18.15 g Tris base (final = 25 mM) 86.4 g Glycine (final = 192 mM) 1200 mL methanol (final = 20%) 4800 mL dH2O 20 MOPS/SDS Running buffer for Bis-Tris Gels For 1 L: 209.2 g MOPS 121.2 g Tris base 20 g SDS 6 g EDTA (free acid) or 7.44 g disodium EDTA 20 NuPAGE Transfer Buffer for Bis-Tris Gels For 500 mL: 40.8 g Bicine 52.32 g Bis-Tris 3 g EDTA (free acid) or 3.8 g disodium EDTA When diluting to 1, include 20% (final) methanol