Western Blot Fluorescence Detection: Step-by-Step Protocol
Fluorescence detection provides quantitative results without the need for film and enables multiplex detection of multiple proteins simultaneously. This comprehensive protocol covers fluorescent secondary antibody selection, scanning techniques, multiplex detection, and image analysis for optimal fluorescence-based western blotting.
Overview
Fluorescence detection uses fluorescently-labeled secondary antibodies and provides several advantages:
- Quantitative: Direct quantification without additional calibration
- Multiplex: Can detect multiple proteins simultaneously with different colors
- No signal fading: Membranes can be re-scanned
- Wide dynamic range: Excellent for quantification
- No film needed: Digital imaging only
This method requires specialized imaging equipment but provides superior quantitative capabilities and multiplex detection options compared to chemiluminescence.
Materials Required
Reagents
- • Fluorescently-labeled secondary antibodies
- • IRDye 680/800 (LI-COR)
- • Alexa Fluor 488/555/647
- • DyLight conjugates
Equipment
- • Fluorescence imaging system
- • LI-COR Odyssey
- • ChemiDoc MP or equivalent
- • Appropriate laser/excitation wavelengths
Fluorescent Secondary Antibody Selection
Choose fluorescent secondary antibodies based on your imaging system and detection needs:
IRDye (LI-COR)
- IRDye 680 (680 nm channel)
- IRDye 800 (800 nm channel)
- Best for LI-COR Odyssey systems
- Excellent for multiplex detection
- Wide dynamic range
Alexa Fluor
- Alexa Fluor 488 (green)
- Alexa Fluor 555 (orange/red)
- Alexa Fluor 647 (far red)
- Compatible with various systems
- Bright and photostable
Selection Guidelines
- Choose wavelengths that don't overlap for multiplex detection
- Ensure compatibility with your imaging system
- Consider signal strength and background levels
- Verify excitation/emission wavelengths match your equipment
Step-by-Step Procedure
Step 1: Secondary Antibody Selection
Use fluorescently-labeled secondary antibodies:
- Common options: IRDye 680/800 (LI-COR), Alexa Fluor 488/555/647, or DyLight conjugates
- Choose wavelengths that don't overlap if detecting multiple proteins
- Ensure compatibility with your imaging system
- Follow manufacturer's dilution recommendations
Step 2: Incubation
Incubate with fluorescent secondary antibody as described in antibody incubation section:
- Protect from light during and after incubation
- Wrap container in aluminum foil or place in dark box
- Prevent photobleaching by avoiding light exposure
- Standard incubation: 1 hour at room temperature
Step 3: Washing
Wash thoroughly with TBST:
- 4-6 washes of 5 minutes each
- Final wash can be with TBS to reduce background fluorescence
- Protect from light during washing
- Ensure complete removal of unbound antibody
Step 4: Scanning
Scan membrane with appropriate laser wavelength:
Scanning Parameters:
- For IRDye: 680 nm and 800 nm channels
- For Alexa Fluor: use appropriate excitation wavelength
- Adjust laser power for optimal signal
- Adjust scan resolution for image quality
- Ensure bands are within linear detection range
Avoid saturation by adjusting laser power. Too high causes saturation, too low misses weak bands.
Step 5: Image Analysis
Use imaging software to quantify band intensity:
- Most systems provide built-in quantification tools
- Ensure all bands are within linear detection range
- Normalize to loading control
- Export data for statistical analysis
Fluorescence detection provides direct quantification without additional calibration steps.
Multiplex Detection
Fluorescence detection allows simultaneous detection of multiple proteins using different colored secondary antibodies:
Multiplex Strategy
- Use secondary antibodies with non-overlapping emission spectra
- Common combinations: IRDye 680 + IRDye 800, or Alexa Fluor 555 + Alexa Fluor 647
- Ensure target proteins are well-separated on gel
- Use appropriate loading controls for each channel
- Verify no cross-talk between channels
Advantages of Multiplex Detection
- Detect multiple proteins from same membrane
- Save time and reagents
- Internal controls on same blot
- Better normalization options
Best Practices
General Guidelines
- Protect from light throughout the process to prevent photobleaching
- Use appropriate laser power - too high causes saturation, too low misses weak bands
- Membranes can be re-scanned if needed (unlike chemiluminescence)
- Quantitative without additional calibration steps
- Store membranes in dark at 4°C if re-scanning later
For Multiplex Detection
- Choose fluorophores with non-overlapping emission spectra
- Verify no cross-talk between channels
- Use appropriate loading controls for each channel
- Ensure target proteins are well-separated
Troubleshooting
Weak Signal
Solutions:
- Increase secondary antibody concentration
- Increase laser power (avoid saturation)
- Check that fluorophore matches imaging system
- Verify antibody is not expired
- Ensure proper primary antibody binding
High Background
Solutions:
- Reduce secondary antibody concentration
- Increase washing stringency
- Use TBS for final wash (without Tween-20)
- Reduce laser power
- Check blocking efficiency
Photobleaching
Solutions:
- Protect from light throughout the process
- Wrap in aluminum foil during incubation
- Store in dark at 4°C
- Scan immediately after washing
- Use photostable fluorophores (IRDye, Alexa Fluor)
Saturation
Solutions:
- Reduce laser power
- Reduce secondary antibody concentration
- Use shorter scan time
- Ensure bands are within linear range