Western Blot for Recombinant Proteins: Complete Guide
Recombinant proteins are proteins expressed from recombinant DNA in host cells such as bacteria, yeast, or mammalian cells. Western blotting is essential for verifying recombinant protein expression, assessing purity, and confirming identity. This comprehensive guide provides optimized protocols for detecting recombinant proteins, including expression verification, sample preparation, detection optimization, and methods to distinguish recombinant proteins from host cell proteins.
Overview
Recombinant proteins are produced in various expression systems, each with unique characteristics:
- Bacterial expression (E. coli): High yield, cost-effective, may lack post-translational modifications
- Yeast expression: Good yield, some post-translational modifications, eukaryotic-like
- Mammalian expression: Full post-translational modifications, lower yield, more expensive
- Insect cell expression: Good yield, some modifications, intermediate cost
Western blotting applications for recombinant proteins:
- Verify protein expression and production
- Assess protein purity after purification
- Confirm protein identity
- Detect protein modifications
- Quantify protein production
- Monitor protein stability
Proper detection methods are essential for accurate assessment of recombinant protein expression and quality.
Expression System Considerations
Bacterial Expression (E. coli)
- High protein yield, often in inclusion bodies
- May require denaturation and refolding
- No post-translational modifications
- Host proteins (E. coli) may contaminate samples
- Use anti-tag antibodies (His-tag, GST-tag) for detection
- May need to detect in insoluble fraction
Yeast Expression
- Good protein yield, often secreted or in soluble fraction
- Some post-translational modifications (glycosylation)
- Host proteins may interfere
- Use tag antibodies or specific antibodies
- Check both intracellular and extracellular fractions
Mammalian Expression
- Full post-translational modifications
- Lower yield, higher cost
- May be secreted or intracellular
- Use specific antibodies or tag antibodies
- Check culture medium for secreted proteins
Sample Preparation
Cell Lysate Preparation
- Harvest cells expressing recombinant protein
- Use appropriate lysis buffer (RIPA, NP-40, or SDS)
- Include protease inhibitors
- For inclusion bodies, use denaturing buffer
- Centrifuge to separate soluble and insoluble fractions
- Check both fractions for protein expression
Purified Recombinant Protein
- If protein is purified, prepare as purified protein sample
- Dilute in appropriate buffer
- Mix with sample buffer
- Heat denature if needed
- Load known amounts for quantification
Tag Detection
- Use anti-tag antibodies (anti-His, anti-GST, anti-FLAG, etc.)
- Tag antibodies are highly specific and sensitive
- Good for detecting recombinant proteins in complex mixtures
- Can use tag antibodies for purification verification
- Verify tag is accessible and not cleaved
Detection Methods
Tag Antibody Detection
- Use anti-tag antibodies for specific detection
- Highly sensitive and specific
- Good for detecting in complex mixtures
- Common tags: His-tag, GST-tag, FLAG-tag, HA-tag
- Optimize antibody concentration for best results
Protein-Specific Antibodies
- Use antibodies specific to the recombinant protein
- Verify antibody recognizes recombinant form
- May need to distinguish from endogenous protein
- Check for cross-reactivity with host proteins
- Optimize detection conditions
Distinguishing from Host Proteins
- Use tag antibodies to specifically detect recombinant protein
- Compare with negative control (untransfected cells)
- Use molecular weight markers to verify size
- Check for expected modifications (glycosylation, etc.)
- Verify expression level is higher than background
Optimization Strategies
Loading Optimization
- Load appropriate amount based on expression level
- For low expression, load more sample
- For high expression, may need to dilute
- Include negative control (untransfected cells)
- Use positive control if available
Detection Optimization
- Optimize antibody concentration through titration
- Use tag antibodies for sensitive detection
- Extend incubation times if needed
- Use sensitive detection methods
- Minimize background from host proteins
Best Practices
- Always include negative control
- Verify tag is present and accessible
- Check both soluble and insoluble fractions
- Document expression conditions
- Compare with expected molecular weight
Troubleshooting
No Expression
- Check induction conditions
- Verify construct and expression system
- Check both soluble and insoluble fractions
- Use sensitive detection methods
- Verify tag is present
Low Expression
- Optimize expression conditions
- Check induction time and temperature
- Increase sample loading
- Use more sensitive detection
- Consider different expression system
High Background
- Improve blocking conditions
- Increase washing steps
- Optimize antibody concentration
- Use tag antibodies for specificity
- Compare with negative control