Western Blot ECL Substrate Problems: Troubleshooting Guide
ECL (Enhanced Chemiluminescence) substrate problems can prevent successful western blot detection. This comprehensive guide helps you troubleshoot ECL substrate issues, including expired substrates, improper mixing, storage problems, and detection failures.
Step 1: Check Substrate Expiration
- Always check expiration date before use
- Expired substrates will not work properly
- Note opening date and use within recommended time
- Store according to manufacturer's instructions
Step 2: Verify Proper Mixing
- Mix ECL components in correct ratio (typically 1:1)
- Mix thoroughly but gently
- Use immediately after mixing (within 5-10 minutes)
- Do not mix too far in advance
- Test on clean membrane to verify activity
Step 3: Check Storage Conditions
- Store ECL components at recommended temperature (usually 4°C)
- Protect from light exposure
- Keep components separate until use
- Avoid freeze-thaw cycles
- Check for contamination or degradation
Common ECL Substrate Problems
No Signal
- Expired substrate
- Improper mixing
- Incorrect storage
- Contamination
Weak Signal
- Partially expired substrate
- Incomplete mixing
- Old working solution
- Insufficient substrate volume
Solutions
- Use fresh, unexpired ECL substrate
- Mix components correctly and thoroughly
- Use working solution immediately after mixing
- Store components properly according to instructions
- Test substrate on clean membrane before use
- Use sufficient substrate volume to cover membrane
- Consider using enhanced ECL substrates for better sensitivity