Western Blot Chemiluminescence Not Working: Fix Guide
When chemiluminescent detection fails, it can halt your entire experiment. This comprehensive troubleshooting guide helps you systematically identify and fix chemiluminescence problems, including substrate issues, HRP activity problems, and imaging system failures.
Step 1: Check ECL Substrate
Substrate Verification
- Check expiration date - expired substrates won't work
- Verify proper storage conditions (usually 4°C, protected from light)
- Test substrate on clean membrane - it should glow
- Ensure components are mixed correctly (typically 1:1 ratio)
- Use working solution immediately after mixing
- Check for contamination or degradation
Step 2: Check HRP Activity
Secondary Antibody HRP
- Verify HRP-conjugated secondary antibody is active
- Check antibody expiration date
- Test with positive control
- Ensure proper storage conditions
HRP Activity Test
- Test HRP activity by applying substrate to secondary antibody alone
- Should see chemiluminescent signal if HRP is active
- If no signal, HRP may be inactive or degraded
Step 3: Check Imaging System
- For X-ray film: Check film is not expired, verify darkroom conditions
- For CCD camera: Check camera settings, focus, and functionality
- Test imaging system with positive control blot
- Verify exposure time is appropriate
- Check for light leaks or contamination
Common Causes
- Expired or degraded ECL substrate
- Improper substrate storage
- Incorrect substrate mixing
- Inactive HRP-conjugated secondary antibody
- Imaging system malfunction
- Incorrect exposure settings
- Membrane drying out
Solutions
- Use fresh, unexpired ECL substrate
- Store substrate components properly
- Mix substrate correctly and use immediately
- Verify HRP-conjugated secondary antibody is active
- Test imaging system with positive control
- Ensure membrane stays wet during detection
- Check all detection protocol steps
- Consider using enhanced ECL substrates