Western Blot Detection Not Working: Troubleshooting Guide
When your detection system fails, it can be frustrating and time-consuming. This comprehensive troubleshooting guide helps you systematically identify and fix detection problems, including substrate issues, imaging system failures, and protocol errors.
Step 1: Check Detection Substrate
Substrate Verification
- Check expiration date of detection substrate
- Verify substrate was stored correctly (according to manufacturer's instructions)
- Test substrate on clean membrane - it should glow
- Ensure substrate components are mixed correctly
- Use fresh substrate prepared immediately before use
Step 2: Check Imaging System
For Chemiluminescence
- Check X-ray film is not expired
- Verify film is handled in darkroom
- Check CCD camera settings and focus
- Verify camera is functioning correctly
- Test with positive control blot
For Fluorescence
- Check laser/excitation source is working
- Verify filter settings are correct
- Check detector sensitivity
- Test with positive control
Step 3: Check Detection Protocol
- Verify substrate was applied evenly to membrane
- Check incubation time is appropriate
- Ensure membrane is not dried out
- Verify detection was performed in dark (for chemiluminescence)
- Check exposure time is appropriate
Common Causes
- Expired or degraded detection substrate
- Improper substrate storage
- Incorrect substrate mixing
- Imaging system malfunction
- Incorrect exposure settings
- Membrane drying out
- Protocol errors
Solutions
- Use fresh, unexpired detection substrate
- Prepare substrate immediately before use
- Test substrate on clean membrane to verify activity
- Check imaging system with positive control
- Verify all detection protocol steps
- Ensure membrane stays wet during detection
- Try alternative detection method if available