Western Blot Secondary Antibody: Complete Guide
Secondary antibody binds to the primary antibody and enables detection through its conjugated enzyme (HRP) or fluorescent tag. Proper selection, dilution, and incubation conditions are crucial for optimal signal-to-noise ratio and specific detection.
Overview
The secondary antibody recognizes and binds to the primary antibody, enabling detection through its conjugated enzyme (HRP) or fluorescent tag. Key points:
- Must be specific to the species of your primary antibody
- Conjugated to detection molecule (HRP, fluorescent dye)
- Proper dilution is critical for signal-to-noise ratio
- Always prepare fresh - never reuse
- Shorter incubation time than primary antibody (1 hour)
Unlike primary antibodies, secondary antibody solutions should always be prepared fresh and never reused, as they degrade and cause high background in subsequent uses.
Secondary Antibody Selection
Proper selection of secondary antibody is critical for successful detection:
Selection Guidelines
- Species matching: Must match primary antibody species (anti-rabbit for rabbit primary, anti-mouse for mouse primary)
- Cross-adsorption: Use highly cross-adsorbed antibodies to minimize cross-reactivity
- Conjugate type: HRP for chemiluminescence, fluorescent dyes (IRDye, Alexa Fluor) for fluorescence
- Multiplex detection: Use secondary antibodies with different emission wavelengths
HRP-Conjugated
- For chemiluminescence detection
- Compatible with ECL substrates
- Most commonly used
- Requires ECL substrate for detection
Fluorescently-Labeled
- For fluorescence detection
- IRDye, Alexa Fluor, or other dyes
- No substrate needed
- Protect from light
Important Notes
- Always use species-specific secondary antibodies
- Highly cross-adsorbed antibodies reduce background
- For multiplex detection, choose dyes with non-overlapping emission spectra
- Ensure compatibility with your detection system
Dilution Guidelines
Secondary antibodies are typically used at higher dilutions than primary antibodies:
HRP-Conjugated
- Typical range: 1:5000 to 1:10000
- Start with manufacturer's recommendation
- Optimize through titration
- Too high concentration causes high background
Fluorescently-Labeled
- Typical range: 1:5000 to 1:15000
- Follow manufacturer's recommendations
- May vary by dye type
- Optimize for your detection system
Dilution Calculation Example
For 1:5000 dilution in 5 mL blocking solution:
- Add 1 μL secondary antibody to 5 mL blocking solution
- For 1:10000 dilution: add 0.5 μL to 5 mL
- Use micropipette for accurate volumes
- Mix gently by inversion or pipetting
- Do not vortex (may cause foaming)
Critical: Always prepare secondary antibody fresh - never reuse solutions. Prepare in blocking solution (same as used for blocking step).
Optimization Strategy
- Start with manufacturer's recommendation: Then optimize through titration
- If background is high: Further dilute (try 1:10000 or 1:15000)
- If signal is weak: Increase concentration (try 1:3000)
- Too high concentration: Causes high background without improving signal
Step-by-Step Procedure
Step 1: Prepare Secondary Antibody Solution
Calculate required volume (5-10 mL for mini-gel):
- Add appropriate volume of blocking solution to clean container
- Using micropipette, add calculated volume of secondary antibody stock
- For 1:5000 dilution in 5 mL: add 1 μL antibody
- Mix gently by pipetting up and down or inverting 5-10 times
- Do not vortex
- Label container with antibody name, dilution, and date
Critical: Always prepare fresh - never reuse secondary antibody solutions. Reused solutions degrade and cause high background.
Step 2: Transfer Membrane to Antibody Solution
After washing primary antibody, briefly drain excess TBST (do not let dry):
- Place membrane protein-side up in secondary antibody solution
- Ensure membrane is completely submerged
- For small membranes: use sealable bag or container
- For larger membranes: use tray or container with enough solution
- Remove any air bubbles by gently tapping or using forceps
Step 3: Incubate with Gentle Agitation
Place container on rocker or shaker set to gentle speed (20-30 rpm):
- Time: 1 hour at room temperature (20-25°C)
- Temperature: Room temperature (not 4°C)
- Agitation: Gentle shaking or rocking
- For fluorescent antibodies: Wrap in aluminum foil or place in dark box
- Monitor: Avoid overheating
Important: Do not extend incubation beyond 1.5 hours - longer incubation increases background without improving signal.
Step 4: Remove Membrane and Proceed to Washing
After incubation, carefully remove membrane:
- Use clean forceps
- Drain excess solution by touching edge to paper towel
- Do not let membrane dry
- Immediately proceed to washing steps
- Discard secondary antibody solution - do not reuse
Optimization Tips
General Guidelines
- Always prepare secondary antibody fresh - do not reuse solutions
- Protect fluorescent antibodies from light during and after incubation
- Use species-specific secondary antibodies to avoid cross-reactivity
- Incubate at room temperature - 4°C incubation is not necessary
- Ensure complete coverage - insufficient solution causes uneven staining
- Do not extend incubation time unnecessarily
- Use gentle agitation - too vigorous shaking can damage membrane
For High Background
- Further dilute secondary antibody (try 1:10000 or 1:15000)
- Reduce incubation time to 45 minutes
- Increase washing stringency
- Use highly cross-adsorbed secondary antibodies
For Weak Signal
- Increase antibody concentration (try 1:3000)
- Ensure proper primary antibody binding
- Check that detection reagents are fresh and working
- Verify antibody compatibility with detection system
Troubleshooting
High Background
Solutions:
- Further dilute secondary antibody (try 1:10000 or 1:15000)
- Reduce incubation time to 45 minutes
- Increase washing stringency
- Use highly cross-adsorbed secondary antibodies
- Check that solution was prepared fresh
Weak Signal
Solutions:
- Increase antibody concentration (try 1:3000)
- Ensure proper primary antibody binding
- Check that detection reagents are fresh and working
- Verify antibody compatibility with detection system
- Check that antibody is not expired
Non-Specific Bands
Solutions:
- Use highly cross-adsorbed secondary antibodies
- Verify species matching (anti-rabbit for rabbit primary)
- Increase washing stringency
- Check for cross-reactivity