Western Blot Housekeeping Proteins: Loading Control Guide
Housekeeping proteins are used as loading controls in western blotting to normalize for variations in sample loading. Choosing the right housekeeping protein is crucial for accurate quantification. This comprehensive guide compares common housekeeping proteins, provides selection criteria, and offers recommendations for different experimental conditions.
Overview
Housekeeping proteins are constitutively expressed proteins used to normalize western blot data. Common housekeeping proteins:
- GAPDH: Glycolytic enzyme (36 kDa)
- β-actin: Cytoskeletal protein (42 kDa)
- α-tubulin: Microtubule protein (50 kDa)
- Vinculin: Adhesion protein (116 kDa)
- Histone H3: Nuclear protein (17 kDa)
The ideal loading control should be stably expressed across all experimental conditions.
GAPDH (36 kDa)
Characteristics
- Glycolytic enzyme, highly expressed
- Present in most cell types
- Good for most applications
- Well-characterized antibodies
Considerations
- May vary under metabolic stress
- May change with glucose levels
- Generally stable for most conditions
β-Actin (42 kDa)
Characteristics
- Cytoskeletal protein, highly expressed
- Present in all cell types
- Most commonly used loading control
- Very stable under most conditions
Considerations
- May vary with cell shape changes
- Generally very stable
- Good for most applications
α-Tubulin (50 kDa)
Characteristics
- Microtubule protein
- Present in most cell types
- Good alternative to actin
- Stable under most conditions
Other Loading Controls
Vinculin (116 kDa)
- Adhesion protein
- Good for large proteins
- Stable expression
Histone H3 (17 kDa)
- Nuclear protein
- Good for nuclear proteins
- Very stable
Selection Guide
How to Choose Loading Control
- For most applications: β-actin or GAPDH
- For nuclear proteins: Histone H3
- For large proteins: Vinculin
- Verify stability: Check that control doesn't vary under your conditions
- Molecular weight: Choose control with different MW than target
- Test multiple: Use multiple controls if unsure