GAPDH Western Blot Loading Control: Complete Protocol
GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) is a highly expressed glycolytic enzyme commonly used as a loading control in western blotting. This comprehensive protocol provides a complete step-by-step guide for detecting GAPDH, including antibody selection, optimal dilution, detection conditions, and troubleshooting tips to ensure reliable loading control results.
Overview
GAPDH is a glycolytic enzyme with a molecular weight of approximately 36 kDa. It is highly expressed in most cell types and is widely used as a loading control because:
- Constitutively expressed in most cells
- Generally stable under most experimental conditions
- Well-characterized antibodies available
- Easy to detect with strong signal
- Molecular weight (36 kDa) is distinct from many target proteins
Note: GAPDH expression may vary under metabolic stress or glucose deprivation conditions. Verify GAPDH stability under your specific experimental conditions.
This protocol provides optimal conditions for detecting GAPDH as a loading control.
Antibody Selection
Recommended Antibodies
- Anti-GAPDH monoclonal antibodies (most common)
- Anti-GAPDH polyclonal antibodies
- Cross-reactive with multiple species (human, mouse, rat)
- Well-validated commercial antibodies available
Antibody Dilution
- Typical dilution: 1:1000 to 1:5000
- Starting point: 1:2000
- Diluent: 3-5% BSA or milk in TBST
- Optimize through titration if needed
Protocol Steps
1. Sample Preparation
- Load 10-30 μg total protein per lane
- Use standard SDS-PAGE gel (10-12%)
- Run gel according to standard protocol
- Transfer to membrane (PVDF or nitrocellulose)
2. Blocking
- Block membrane in 5% milk or BSA in TBST
- Block for 1 hour at room temperature
- Or block overnight at 4°C
3. Primary Antibody Incubation
- Dilute GAPDH antibody 1:2000 in blocking buffer
- Incubate for 1 hour at room temperature
- Or incubate overnight at 4°C (more sensitive)
- Use gentle rocking during incubation
4. Washing
- Wash 3-4 times with TBST
- 5 minutes per wash
- Use sufficient wash buffer volume
5. Secondary Antibody
- Dilute HRP-conjugated secondary antibody 1:5000
- Incubate for 1 hour at room temperature
- Wash 3-4 times with TBST
6. Detection
- Use chemiluminescent detection
- Short exposure time (usually 1-5 seconds)
- GAPDH signal should be strong and clear
Optimization Tips
- If signal is too strong: Reduce antibody dilution to 1:5000
- If signal is weak: Increase to 1:1000 or extend incubation
- For high background: Increase washing stringency
- Ensure equal loading across all lanes
- Verify GAPDH stability under your experimental conditions
- Consider alternative loading control if GAPDH varies under your conditions
Troubleshooting
No Signal
- Check antibody expiration date
- Increase antibody concentration
- Extend incubation time
- Verify sample loading
Signal Too Strong
- Reduce antibody dilution
- Reduce exposure time
- Dilute samples if overloading
Variable Signal
- Check for equal sample loading
- Verify GAPDH stability under conditions
- Consider alternative loading control (e.g., β-actin, tubulin)
- Check for metabolic stress or glucose deprivation