The western blot is one of the most widely used techniques in molecular biology, yet it’s also one of the most failure-prone. Faint bands, smeary lanes, high background — every lab has war stories. The good news: most issues come from a handful of well-understood mistakes.
1. Sample preparation
Lyse cells in an appropriate buffer (RIPA for whole-cell, NP-40 for cytoplasmic, nuclear extraction kits for nuclear proteins). Always add protease inhibitors fresh, and phosphatase inhibitors if you’re probing phospho-proteins. Quantify with BCA or Bradford — eyeballing it is the fastest path to inconsistent loading. Load 10–30 µg per lane. Boil samples in Laemmli buffer at 95 °C for 5 minutes, except membrane proteins (warm at 37–70 °C to avoid aggregation).
2. SDS-PAGE
Choose gel percentage by target size: 6–8% for proteins above 100 kDa, 10% for 30–100 kDa, 12–15% for under 30 kDa. Run at 80 V through stacking gel, then 120 V through resolving gel.
3. Transfer
Wet (tank) transfer is the most reliable for large proteins. Semi-dry is faster but inconsistent above 150 kDa. Use PVDF (activate first in methanol) for high sensitivity, or nitrocellulose for general work. After transfer, stain with Ponceau S to confirm even loading and successful transfer before committing antibodies. This single step catches half of all “western didn’t work” problems.
4. Blocking
Block in 5% non-fat dry milk in TBST for 1 hour at room temperature. Switch to 5% BSA when probing phospho-proteins — milk contains casein, which is itself phosphorylated and creates background.
5. Primary antibody
Dilute primary in blocking buffer per manufacturer’s recommendation (typically 1:500 to 1:5,000). Incubate overnight at 4 °C with gentle rocking. This long, cold incubation gives consistently cleaner results than 1 hour at room temperature. Wash 3 × 10 minutes in TBST.
6. Secondary antibody and detection
Apply HRP-conjugated secondary (1:5,000–1:20,000) in blocking buffer for 1 hour at room temperature. Wash 3 × 10 minutes. Develop using ECL substrate and image with a CCD-based system or film.
7. Loading controls
Probe a loading control: GAPDH, β-actin, or β-tubulin for whole-cell lysates; lamin B1 or histone H3 for nuclear fractions. For quantification, total protein normalization (Ponceau or stain-free gels) is now preferred — many journals require it because housekeeping proteins aren’t always invariant.
Common problems and quick fixes
- No signal: Check transfer with Ponceau, verify antibody dilution, increase exposure time.
- High background: Increase wash duration, switch to BSA, dilute antibody further.
- Multiple bands: Could be specific (isoforms, modifications) or non-specific. Validate with knockout/knockdown.
- Smeary bands: Sample degradation — add fresh inhibitors, keep on ice, avoid freeze-thaw cycles.
Document every step, run controls, and the rest is iteration.
