Western Blot Gel Percentage Guide: How to Choose
Choosing the correct gel percentage is crucial for optimal protein separation in western blotting. The gel percentage determines the pore size and affects how proteins migrate through the gel. This comprehensive guide provides expert recommendations for selecting the appropriate gel percentage based on protein size, with detailed guidelines and optimization tips.
Overview
Gel percentage refers to the concentration of polyacrylamide in the gel, which determines pore size and separation resolution:
- Higher percentage: Smaller pores, better for small proteins
- Lower percentage: Larger pores, better for large proteins
- Gradient gels: Varying percentage, good for wide size range
The optimal gel percentage depends on your target protein's molecular weight.
Gel Percentage by Protein Size
| Protein Size | Recommended Gel % | Alternative |
|---|---|---|
| <20 kDa | 15-20% | 12-15% |
| 20-50 kDa | 12-15% | 10-12% |
| 50-100 kDa | 10-12% | 8-10% |
| 100-200 kDa | 7-10% | 6-8% |
| >200 kDa | 4-7% | Gradient gel |
Gel Types
Single Percentage Gels
- Uniform polyacrylamide concentration throughout
- Good for proteins within narrow size range
- Simpler to prepare
- Most commonly used
Gradient Gels
- Varying polyacrylamide concentration (e.g., 4-20%)
- Good for wide protein size range
- Better resolution across size range
- More complex to prepare
Selection Guide
Quick Selection Guide
- Small proteins (<20 kDa): Use 15-20% gel
- Medium proteins (20-100 kDa): Use 10-12% gel (most common)
- Large proteins (>100 kDa): Use 7-10% gel
- Very large proteins (>200 kDa): Use 4-7% gel or gradient
- Multiple proteins: Use gradient gel or compromise percentage
Optimization Tips
- Start with standard percentage for your protein size
- Test different percentages if resolution is poor
- Use gradient gels for wide size ranges
- Consider stacking gel percentage (typically 4-5%)
- Optimize running conditions with gel percentage