- Prepare bis-tris gels or use precast TGX gels (Bio-Rad).
- Dilute samples appropriately in NuPAGE sample buffer (for bis-tris) or Laemmli buffer (for TGX).
- Boil samples 5 minutes at 100ºC in heating block.
- Load 10-50 µL protein sample per well.
- Add appropriate running buffer to tank.
- Run gel 200 V, 0.5 hour.
- Remove gel from glass or plastic plates, cut off stacking gel.
- Soak 6×9 cm nitrocellulose membrane in transfer buffer appropriate for gel system (use forceps).
- 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.
- Layer gel on top of paper, roll out bubbles.
- Carefully place membrane on top of gel.
- Layer another soaked blotting paper square on top, roll out bubbles.
- Add sponge.
- Close sandwich, place in transfer unit, with membrane side closest to positive electrode (red).
- Transfer 75 V, 90 min.
- After transfer, gels and blots may be stained.
- 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.
- To stain membrane: Shake in 5 mL Ponceau S (recyclable) for 5 minutes.
- Rinse thoroughly with water to visualize pink protein bands.
- Cut or mark gel bands as necessary now; stain will disappear during blocking.
- Block membrane by shaking in 5 mL 5% skim milk powder in TBST, 1 hour.
- Replace blocking solution with a similar solution containing an appropriate dilution of primary antibody.
- Shake for 1-3 hours at RT OR shake at 4°C overnight (covered).
- Remove and save antibody solution (preserve with 0.02% sodium azide).
- Rinse blot twice with TBST.
- Wash three times for five minutes in TBST.
- Prepare secondary antibody dilution in 5% skim milk powder in TBST.
- Add secondary antibody solution, incubate with shaking 1 hour at RT.
- Discard antibody solution, and rinse blot twice with TBST.
- Wash five times for five minutes in TBST.
- Cover with ECL+ reagent (25 µL Solution B + 975 µL Solution A per blot).
- Transfer to GelDoc or film cassette (in Saran wrap) for imaging.
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
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
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