Western Blot Antibody Dilution Guide: How to Dilute
Proper antibody dilution is crucial for successful western blotting. Too high concentration can cause high background, while too low concentration can result in weak or no signal. This comprehensive guide provides expert methods for diluting primary and secondary antibodies, including titration protocols, optimization strategies, and troubleshooting tips.
Primary Antibody Dilution
Typical Dilution Range
- Most primary antibodies: 1:500 to 1:5000
- High-affinity antibodies: 1:1000 to 1:10000
- Low-affinity antibodies: 1:100 to 1:1000
- Start with manufacturer's recommendation
- Optimize through titration
Dilution Buffer
- Dilute in blocking buffer (3-5% BSA or milk in TBST)
- Include 0.1% Tween-20 in buffer
- Use fresh buffer for each experiment
- Keep buffer cold if storing diluted antibody
Secondary Antibody Dilution
Typical Dilution Range
- HRP-conjugated secondary: 1:5000 to 1:20000
- Fluorescent secondary: 1:5000 to 1:10000
- Start with 1:5000 for most applications
- Optimize based on signal and background
- Higher dilutions reduce background
Antibody Titration Protocol
Titration Steps
- Prepare serial dilutions (1:100, 1:500, 1:1000, 1:2000, 1:5000)
- Test each dilution on the same blot or replicate blots
- Compare signal strength and background
- Select dilution with best signal-to-background ratio
- Document optimal dilution for future use
Optimization Tips
For Strong Signal
- Use higher antibody concentration
- Extend incubation time
- Use enhanced detection substrates
For Low Background
- Use lower antibody concentration
- Increase washing stringency
- Optimize blocking conditions
Troubleshooting
- No signal: Try higher concentration (1:100 to 1:500)
- Weak signal: Increase concentration or extend incubation
- High background: Reduce concentration or increase washing
- Non-specific bands: Reduce concentration and improve blocking