Western Blot Chemiluminescence Detection: Step-by-Step Protocol
Chemiluminescence is the most sensitive and widely used detection method for western blotting. This detailed protocol covers ECL substrate preparation, membrane incubation, image capture techniques, exposure optimization, and troubleshooting to achieve optimal chemiluminescent detection results.
Overview
Chemiluminescence detection uses ECL (Enhanced Chemiluminescence) substrates that produce light when catalyzed by HRP-conjugated secondary antibodies. This method offers:
- Highest sensitivity: Can detect picogram levels of protein
- Excellent dynamic range: 4-5 orders of magnitude
- Widely available: Most common detection method
- Cost-effective: Relatively inexpensive substrates
- Signal fading: Signal peaks within minutes and then fades
The key to successful chemiluminescence detection is proper substrate preparation, optimal exposure timing, and capturing images within the linear detection range.
Materials Required
Reagents
- • ECL substrate (commercial kits)
- • Pierce ECL, SuperSignal, or equivalent
- • Enhanced ECL substrates (for weak signals)
Equipment
- • X-ray film or CCD imaging system
- • Plastic wrap or transparency film
- • Darkroom or imaging chamber
- • Film developer (if using X-ray film)
Step-by-Step Procedure
Step 1: Prepare ECL Substrate
Mix ECL substrate components according to manufacturer's instructions:
- Most ECL kits have two components (luminol and peroxide)
- Mix equal volumes (1:1 ratio) just before use
- Mix gently by pipetting or inverting
- Use immediately after mixing
- Do not store mixed substrate
Important: Mix substrate components just before use. The reaction begins immediately upon mixing, and signal peaks within minutes.
Step 2: Incubate Membrane
Place membrane protein-side up on a clean surface:
- Pipette ECL substrate onto membrane
- Ensure complete coverage of the membrane
- Incubate for 1-5 minutes at room temperature
- Longer incubation (up to 5 minutes) may increase signal
- Monitor for background development
For very weak signals, extended incubation (up to 10 minutes) or enhanced ECL substrates may help.
Step 3: Remove Excess Substrate
After incubation:
- Drain excess substrate by tilting membrane
- Do not let membrane dry
- Wrap in plastic wrap or place in imaging cassette
- Ensure no air bubbles between membrane and wrap
- Work quickly - signal peaks within minutes
Step 4: Image Capture
Capture image using your preferred method:
For X-ray Film:
- Expose film for 1 second to 10 minutes depending on signal strength
- Start with short exposure (1-5 seconds) and adjust
- Develop film according to manufacturer's instructions
- Take multiple exposures at different times
For CCD Camera:
- Place in imaging system
- Capture image
- Adjust exposure time to avoid saturation
- Ensure bands are within linear detection range
Step 5: Multiple Exposures
Take multiple exposures at different times:
- Capture both strong and weak bands within linear range
- Document exposure time for each image
- Use shortest exposure that shows all bands
- Avoid overexposure (saturation)
Multiple exposures ensure you capture both strong and weak bands within the linear detection range.
Exposure Optimization
Optimal exposure is critical for accurate quantification and clear visualization:
Exposure Guidelines
- Start short: Begin with 1-5 second exposure
- Adjust incrementally: Increase or decrease based on signal strength
- Linear range: Ensure all bands are within linear detection range
- Avoid saturation: Overexposed bands cannot be quantified accurately
- Document time: Record exposure time for each image
Signal Timing
- Peak signal: Occurs within 1-5 minutes after substrate addition
- Fading: Signal fades over time (minutes to hours)
- Work quickly: Capture images soon after substrate addition
- Re-expose: Can add more substrate and re-expose if needed
Best Practices
General Guidelines
- Work quickly after adding substrate - signal peaks within minutes
- Keep membrane moist - drying reduces signal
- Avoid light exposure before imaging
- Use fresh ECL substrate - old substrate may not work
- Store ECL components according to manufacturer's instructions
For Weak Signals
- Try extended incubation (up to 10 minutes)
- Use enhanced ECL substrates
- Increase secondary antibody concentration
- Extend primary antibody incubation time
- Check that all reagents are fresh
Troubleshooting
No Signal
Possible causes:
- ECL substrate not mixed correctly or expired
- Secondary antibody not bound properly
- Signal faded before imaging
- Exposure time too short
- Check that HRP-conjugated secondary antibody was used
Weak Signal
Solutions:
- Extend substrate incubation time (up to 10 minutes)
- Use enhanced ECL substrates
- Increase secondary antibody concentration
- Extend exposure time
- Check that substrate is fresh
High Background
Solutions:
- Reduce substrate incubation time
- Reduce secondary antibody concentration
- Increase washing stringency
- Reduce exposure time
- Check blocking efficiency
Signal Faded
Solutions:
- Add fresh substrate and re-expose
- Work more quickly after substrate addition
- Capture images immediately after substrate incubation
- Use enhanced ECL substrates for longer signal duration