Before you start
- Cut membrane and filter paper to gel size; activate PVDF in methanol for 30 s.
- Use BSA for blocking and antibodies with phospho-specific antibodies (milk contains phosphoproteins).
Reagents and materials
Totals are for 2 membranes (+10% extra). Per-membrane (mini) amounts are in the middle column.
| Reagent | Per membrane (mini) | Total |
|---|---|---|
| Transfer buffer (1× Tris-glycine + 20% methanol)wet tank | 1000 mL | 1000 mL |
| Methanol (PVDF activation) | 10 mL | 10 mL |
| Ponceau S (0.1% in 5% acetic acid) | 10 mL | 10 mL |
| Blocking buffer (5% milk or 5% BSA in TBST) | 15 mL | 15 mL |
| Primary antibody in blocking buffer | 5 mL | 5 mL |
| HRP secondary antibody (1:5,000–1:20,000) | 10 mL | 10 mL |
| TBST (washes) | 150 mL | 150 mL |
| ECL substrate | 1 mL | 1 mL |
Buffers and recipes
Transfer buffer per 1 L
| Tris base (25 mM) | 3.03 g |
| Glycine (192 mM) | 14.4 g |
| Methanol (20%) | 200 mL |
| Ultrapure water | to 1 L |
Chill before use. Reduce methanol to 10% and add 0.01–0.05% SDS for large proteins.
10× TBS (dilute to 1×, add Tween-20 for TBST) per 1 L
| Tris base (200 mM) | 24.2 g |
| NaCl (1.37 M) | 80 g |
| Ultrapure water | to 1 L |
Adjust to pH 7.6 with HCl. For 1× TBST: 100 mL 10× TBS + 900 mL water + 1 mL Tween-20 (0.1%).
Equipment
- Wet or semi-dry transfer system
- PVDF (0.45 or 0.2 µm) or nitrocellulose membrane
- Filter paper and sponges
- Rocker
- Chemiluminescence imager
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Equilibrate
Soak gel, sponges, filter papers and membrane in cold transfer buffer.
Build the sandwich
Black side (−) → sponge → filter paper → gel → membrane → filter paper → sponge → red side (+). Roll out bubbles.
Transfer
Wet transfer at 100 V for 60–90 min on ice or with a cold pack (or 30 V overnight at 4 °C).
Check transfer
Stain with Ponceau S for 1–2 min, image, then destain with TBST.
Block
Block 1 h at room temperature with rocking.
Primary antibody
Incubate overnight at 4 °C with rocking.
Wash
Wash 3 × 5–10 min in TBST.
Secondary antibody
Incubate with HRP secondary for 1 h at room temperature.
Wash
Wash 3 × 10 min in TBST.
Detect
Incubate with ECL for 1–5 min and image with several exposures, avoiding saturation.
Quantify
Quantify bands in the linear range and normalise to a loading control or total-protein stain.
Critical points
- Choose 0.2 µm membranes for proteins under ~20 kDa.
- Use total-protein normalisation or validated loading controls that do not change with treatment.
Troubleshooting
| Problem | What to check or change |
|---|---|
| No bands | Check transfer (Ponceau), antibody species and dilution, ECL freshness. |
| High background | Block longer, dilute antibodies more, wash more, switch blocking agent. |
| Extra bands | Titrate primary, add inhibitors to lysis, check antibody specificity with knockout samples. |
Safety
Methanol is toxic and flammable. Keep the transfer cold to avoid overheating.
Further reading
- Towbin H et al. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. PNAS (1979).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.