2. Dilute samples appropriately in NuPAGE sample buffer (for bis-tris) or Laemmli buffer (for TGX).
3. Boil samples 5 minutes at 100ºC in heating block.
4. Load 10-50 µL 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.
1. Soak 6×9 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 (7×10 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.
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.