Western Blot Antibody Incubation: Complete Guide
Proper antibody incubation is essential for successful western blotting. This comprehensive guide covers primary and secondary antibody incubation, dilution optimization, incubation conditions, and best practices to achieve optimal signal-to-noise ratio and specific detection.
Overview
Antibody incubation involves two sequential steps:
- Primary antibody: Binds specifically to your target protein
- Secondary antibody: Binds to the primary antibody and enables detection
Both steps require proper dilution, incubation conditions, and thorough washing. Key factors affecting success:
- Antibody dilution (concentration)
- Incubation time and temperature
- Blocking solution compatibility
- Washing stringency
- Antibody specificity and quality
Primary Antibody Incubation
The primary antibody specifically recognizes and binds to your target protein. Proper incubation is critical for specific detection. See our detailed guide: Primary Antibody Protocol.
Key Points
- Dilution: Monoclonal: 1:1000-1:5000, Polyclonal: 1:500-1:2000
- Incubation: Overnight at 4°C (recommended) or 1-2 hours at room temperature
- Solution: Dilute in blocking solution (same as used for blocking)
- Volume: 5-10 mL for mini-gel, ensure complete coverage
- Reuse: Can be reused 2-3 times if stored properly with sodium azide
Incubation Conditions
Overnight at 4°C (Recommended):
- Best signal-to-noise ratio
- Most sensitive detection
- 12-16 hours with gentle shaking
Room Temperature:
- Faster (1-2 hours)
- Convenient for same-day results
- May have slightly higher background
Secondary Antibody Incubation
The secondary antibody binds to the primary antibody and enables detection through its conjugated enzyme (HRP) or fluorescent tag. See our detailed guide: Secondary Antibody Protocol.
Key Points
- Selection: Must match primary antibody species (anti-rabbit for rabbit primary)
- Dilution: HRP-conjugated: 1:5000-1:10000, Fluorescent: 1:5000-1:15000
- Incubation: 1 hour at room temperature with gentle shaking
- Volume: 5-10 mL for mini-gel
- Reuse: Do NOT reuse - always prepare fresh
Important Notes
- Always prepare secondary antibody fresh - never reuse
- Protect fluorescent antibodies from light
- Use species-specific secondary antibodies
- Do not extend incubation beyond 1.5 hours
Washing Steps
Thorough washing removes unbound antibodies and reduces background signal. Washing is performed after both primary and secondary antibody incubations:
After Primary Antibody
- 4-6 washes with TBST, 5 minutes each
- Use fresh TBST for each wash
- Gentle shaking (20-30 rpm)
- Ensure complete coverage
After Secondary Antibody
- 4-6 washes with TBST, 5 minutes each
- More stringent washing may be needed
- Final wash can be with TBS (without Tween-20) if needed
- Proceed immediately to detection
Washing Best Practices
- Always use fresh TBST for each wash
- Ensure adequate volume (20-50 mL per wash)
- Maintain gentle agitation
- Do not let membrane dry between washes
- Do not over-wash (can reduce specific signal)
Optimization Strategies
For Weak Signal
- Increase primary antibody concentration (try 1:500 for polyclonal, 1:500 for monoclonal)
- Extend primary antibody incubation time
- Use overnight incubation at 4°C
- Verify antibody specificity with positive control
- Check that detection reagents are fresh
For High Background
- Decrease antibody concentration
- Increase blocking time
- Try different blocking solution (BSA instead of milk)
- Increase washing stringency (more washes or longer time)
- For secondary antibody: Further dilute (1:10000 or 1:15000)
For Uneven Signal
- Ensure adequate volume - membrane should float freely
- Remove air bubbles
- Maintain consistent agitation
- Check that membrane is not folded or creased
Troubleshooting
No Signal
Possible causes:
- Antibody too dilute - increase concentration
- Insufficient incubation time - extend time
- Antibody not specific or expired
- Protein not transferred - check with Ponceau S
- Detection reagents expired or not working
High Background
Solutions:
- Decrease antibody concentration
- Increase blocking time
- Try different blocking solution
- Increase washing stringency
- Use highly cross-adsorbed secondary antibodies
Non-Specific Bands
Solutions:
- Verify antibody specificity
- Use blocking solution appropriate for your application
- Increase washing stringency
- Check for cross-reactivity