How to Choose Western Blot Membrane: Complete Guide
Choosing the right membrane is essential for successful western blotting. The membrane type affects protein binding capacity, detection sensitivity, and experimental workflow. This comprehensive guide provides expert criteria for selecting the best membrane, including comparisons of PVDF and nitrocellulose, pore size selection, and recommendations for different applications.
Membrane Types
PVDF (Polyvinylidene Difluoride)
- High protein binding capacity (150-200 μg/cm²)
- Excellent mechanical strength
- Requires methanol activation
- Compatible with chemiluminescence and fluorescence
- Can be stripped and reprobed multiple times
- More expensive than nitrocellulose
Nitrocellulose
- Good protein binding capacity (80-100 μg/cm²)
- Hydrophilic, wets easily
- No activation required
- Compatible with chemiluminescence and fluorescence
- Less expensive than PVDF
- Less durable than PVDF
PVDF vs Nitrocellulose Comparison
| Feature | PVDF | Nitrocellulose |
|---|---|---|
| Binding Capacity | High | Good |
| Mechanical Strength | Excellent | Good |
| Activation Required | Yes (methanol) | No |
| Cost | Higher | Lower |
| Stripping/Reprobing | Excellent | Good |
Pore Size Selection
0.2 μm Pore Size
- For small proteins (<20 kDa)
- Prevents small proteins from passing through
- Higher binding capacity
0.45 μm Pore Size
- For most proteins (standard)
- Good for proteins 20-200 kDa
- Most commonly used
Selection Criteria
Choose PVDF When:
- Detecting low abundance proteins
- Need maximum binding capacity
- Plan to strip and reprobe
- Need durable membrane
- Long-term storage required
Choose Nitrocellulose When:
- Routine western blotting
- Cost is a concern
- Want easy-to-use membrane
- Detecting moderate to high abundance proteins
- Single detection (no reprobing)
General Recommendations
- For most applications: Nitrocellulose 0.45 μm is sufficient
- For low abundance: PVDF 0.2 μm offers better binding
- For small proteins: Use 0.2 μm pore size
- For reprobing: PVDF is more durable
- For beginners: Nitrocellulose is easier to use