Western Blot for Tissue Extracts: Complete Guide
Tissue extracts provide a comprehensive view of protein expression in intact tissues, reflecting the in vivo state more accurately than cell culture. Preparing tissue extracts for western blot requires specialized homogenization and extraction methods due to tissue complexity and heterogeneity. This comprehensive guide provides optimized protocols for tissue extract preparation, including homogenization techniques, buffer selection, protein extraction, and optimization strategies for different tissue types.
Overview
Tissue extracts are prepared from intact tissues and contain proteins from all cell types present in the tissue. Key advantages:
- Reflect in vivo protein expression and modifications
- Include proteins from multiple cell types
- Represent tissue-specific protein patterns
- Allow study of tissue-specific responses
- Provide physiological context
Key challenges in tissue extract preparation:
- Tissue heterogeneity requires thorough homogenization
- High protein and DNA content can cause viscosity
- Presence of connective tissue and extracellular matrix
- Variable protein content between tissue types
- Risk of protein degradation during processing
- Need for efficient extraction from complex structures
Proper homogenization and extraction methods are essential for obtaining high-quality tissue extracts.
Tissue Preparation
Fresh Tissue
- Process immediately after collection when possible
- Rinse with cold PBS to remove blood and debris
- Cut into small pieces (2-5 mm) for easier homogenization
- Weigh tissue to normalize protein content
- Keep tissue cold (4°C or on ice) throughout preparation
Frozen Tissue
- Keep tissue frozen until ready to process
- Pulverize frozen tissue using mortar and pestle (pre-cooled in liquid nitrogen)
- Or cut frozen tissue into small pieces while frozen
- Add lysis buffer immediately after pulverization
- Avoid thawing before adding lysis buffer
Tissue Weight and Buffer Ratio
- Use 50-100 mg tissue per 500-1000 μL lysis buffer
- Adjust ratio based on tissue type and protein content
- Weigh tissue accurately for normalization
- Consider tissue density and composition
Homogenization Methods
Mechanical Homogenization
- Dounce homogenizer: Gentle, good for soft tissues (brain, liver)
- Polytron homogenizer: Powerful, good for tough tissues (muscle, heart)
- Bead beater: Effective for small samples
- Homogenize on ice to prevent heating
- Use multiple passes if needed (10-20 strokes)
- Monitor homogenization to avoid over-processing
Sonication
- Use probe sonicator or bath sonicator
- Sonicate on ice in short bursts (10-30 seconds)
- Allow cooling between bursts
- Monitor to avoid foaming and heating
- Good for small samples or as supplement to mechanical homogenization
Grinding Methods
- Mortar and pestle: For frozen tissue (pre-cooled in liquid nitrogen)
- Mechanical grinder: For larger samples
- Keep tissue frozen during grinding
- Add lysis buffer immediately after grinding
- Effective for very tough tissues
Protein Extraction
Extraction Protocol
- Homogenize tissue in lysis buffer (RIPA or appropriate buffer)
- Incubate on ice for 30-60 minutes with occasional vortexing
- Centrifuge at 10,000-15,000 × g for 15-20 minutes at 4°C
- Collect supernatant (soluble proteins)
- If needed, re-extract pellet with harsher buffer
- Determine protein concentration
- Prepare samples for western blot
Lysis Buffer Selection
- RIPA buffer: Most common, effective for most tissues
- NP-40 buffer: Milder, for sensitive proteins
- SDS buffer: Harsher, for difficult extractions
- Include complete protease inhibitor cocktail
- Add phosphatase inhibitors if detecting phosphoproteins
Viscosity Reduction
- Add DNAse (10-50 U/mL) to reduce DNA-induced viscosity
- Dilute samples if too viscous
- Centrifuge longer or at higher speed
- Filter through 0.45 μm filter if needed
- Consider using less tissue per volume of buffer
Tissue-Specific Considerations
Brain Tissue
- High lipid content - may need delipidation
- Soft tissue - use gentle homogenization
- High protein content - may need dilution
- Use RIPA buffer with protease inhibitors
Muscle Tissue
- Tough tissue - requires powerful homogenization
- High protein content - use appropriate buffer volume
- May need extended homogenization time
- Consider using Polytron or similar powerful homogenizer
Liver Tissue
- High protease activity - use strong protease inhibitors
- Process quickly to prevent degradation
- High protein content - may need dilution
- Use RIPA buffer with complete inhibitor cocktail
Heart Tissue
- Tough tissue - requires powerful homogenization
- May need extended homogenization
- Use appropriate buffer volume
- Consider grinding methods for frozen tissue
Optimization Tips
Homogenization Optimization
- Choose appropriate homogenization method for tissue type
- Optimize homogenization time and intensity
- Keep samples cold during homogenization
- Monitor homogenization to avoid over-processing
- Test different methods to find optimal
Sample Quality
- Process samples quickly to prevent degradation
- Include complete protease inhibitor cocktail
- Keep all steps cold (4°C or on ice)
- Minimize freeze-thaw cycles
- Store samples at -80°C with inhibitors
Normalization
- Normalize to tissue weight (mg tissue per lane)
- Or normalize to total protein content
- Use loading controls (GAPDH, β-actin)
- Consider tissue-specific loading controls
Troubleshooting
High Viscosity
- Add DNAse to reduce DNA content
- Dilute samples with lysis buffer
- Centrifuge longer or at higher speed
- Use less tissue per volume of buffer
Low Protein Yield
- Increase homogenization time or intensity
- Use harsher lysis buffer (add more SDS)
- Re-extract pellet with stronger buffer
- Check for incomplete tissue disruption
Protein Degradation
- Include complete protease inhibitor cocktail
- Process samples more quickly
- Keep samples cold throughout
- Use fresh inhibitors