Western Blot Sample Loading Amount: How Much to Load
Determining the correct sample loading amount is crucial for successful western blotting. Too little protein may result in weak or no signal, while too much can cause overloading artifacts and poor resolution. This comprehensive guide provides expert recommendations for sample loading amounts, optimization strategies, and troubleshooting tips for different sample types and protein abundances.
Standard Loading Amounts
Recommended Loading Amounts
- Standard proteins: 10-30 μg total protein per lane
- Low abundance proteins: 50-100 μg per lane
- High abundance proteins: 5-10 μg per lane
- Very low abundance: 100-200 μg per lane (may need concentration)
- Starting point: 20-30 μg for most applications
Protein Abundance Considerations
High Abundance Proteins
- Load 5-10 μg per lane
- May need to dilute samples
- Watch for overloading artifacts
- Examples: Actin, GAPDH, tubulin
Low Abundance Proteins
- Load 50-100 μg per lane
- May need sample concentration
- Use sensitive detection methods
- Examples: Transcription factors, signaling proteins
Optimization Strategies
How to Optimize Loading Amount
- Start with standard amount (20-30 μg)
- If no signal: Increase to 50-100 μg
- If overloading: Reduce to 10-20 μg
- Test different amounts systematically
- Document optimal amount for future use
Troubleshooting
No Signal or Weak Signal
- Increase loading amount to 50-100 μg
- Concentrate samples if needed
- Verify protein concentration
- Check for protein degradation
Overloading Artifacts
- Reduce loading amount to 10-20 μg
- Dilute samples before loading
- Check for distorted bands
- Verify protein concentration
Sample Type Guidelines
- Cell lysates: 10-30 μg (standard), 50-100 μg (low abundance)
- Tissue extracts: 20-50 μg (may need more for tough tissues)
- Purified proteins: 0.1-1 μg (much less needed)
- Serum/plasma: 5-20 μL (diluted 1:10 to 1:50)
- Recombinant proteins: 0.1-5 μg depending on expression level