Western Blot for Post-Translational Modifications: Complete Guide
Post-translational modifications (PTMs) are chemical modifications that occur after protein synthesis, regulating protein function, localization, and interactions. Detecting PTMs in western blot requires specialized protocols to preserve modification status and use modification-specific antibodies. This comprehensive guide provides optimized methods for detecting various PTMs including phosphorylation, acetylation, methylation, ubiquitination, and other modifications.
Overview
Post-translational modifications are essential for regulating protein function and include:
- Phosphorylation: Addition of phosphate groups (most common PTM)
- Acetylation: Addition of acetyl groups (lysine acetylation)
- Methylation: Addition of methyl groups (lysine, arginine methylation)
- Ubiquitination: Addition of ubiquitin (protein degradation signal)
- SUMOylation: Addition of SUMO (small ubiquitin-like modifier)
- Glycosylation: Addition of sugar moieties
- Other modifications: Hydroxylation, nitration, citrullination, etc.
Key considerations for PTM detection:
- PTMs are often labile and can be lost during sample preparation
- Modification-specific antibodies are required
- Sample preparation must preserve modification status
- Blocking agents must not interfere with modification detection
- PTMs are often substoichiometric (low modification levels)
- Multiple modifications can occur on the same protein
Proper sample handling, modification-specific protocols, and appropriate antibodies are essential for successful PTM detection.
Phosphorylation Detection
Sample Preparation
- Include phosphatase inhibitors in lysis buffer (sodium orthovanadate, sodium fluoride, β-glycerophosphate)
- Process samples quickly at 4°C to prevent dephosphorylation
- Use RIPA buffer with complete phosphatase inhibitor cocktail
- Avoid freeze-thaw cycles that can affect phosphorylation
- Store samples at -80°C with inhibitors if not processing immediately
Blocking Strategy
- Never use milk: Milk contains active phosphatases that dephosphorylate proteins
- Use BSA: Block with 3-5% BSA in TBST
- Phosphatase-free blocking: Use commercially available phosphatase-free blocking solutions
- Block for 1-2 hours at room temperature
Antibody Selection
- Use phospho-specific antibodies that recognize phosphorylated residues
- Verify antibody specificity for specific phosphorylation sites
- Test with positive and negative controls
- Consider site-specific vs pan-phospho antibodies
- Optimize antibody concentration (typically 1:500 to 1:2000)
Acetylation Detection
Sample Preparation
- Include HDAC (histone deacetylase) inhibitors in lysis buffer (trichostatin A, sodium butyrate)
- Process samples quickly to prevent deacetylation
- Use appropriate lysis buffer (RIPA or nuclear extraction buffer)
- Keep samples cold during preparation
Antibody and Detection
- Use acetylation-specific antibodies (anti-acetyl-lysine)
- For histones, use histone-specific acetylation antibodies
- Optimize antibody concentration through titration
- Standard blocking and detection protocols usually work
Methylation Detection
Sample Preparation
- Methylation is generally stable and doesn't require special inhibitors
- Use standard lysis buffers
- For histones, may require acid extraction
- Process samples using standard protocols
Antibody Selection
- Use methylation-specific antibodies (mono-, di-, tri-methylation specific)
- For histones, use site-specific methylation antibodies
- Verify antibody specificity and modification state recognition
- Optimize antibody concentration
Ubiquitination Detection
Sample Preparation
- Include deubiquitinase (DUB) inhibitors in lysis buffer (N-ethylmaleimide, PR-619)
- Process samples quickly to prevent deubiquitination
- Use denaturing lysis buffer to preserve ubiquitination
- Heat samples at 95°C immediately after lysis
- Avoid reducing agents that can affect ubiquitin chains
Detection Methods
- Use anti-ubiquitin antibodies (mono-ubiquitin or poly-ubiquitin specific)
- Consider chain-specific antibodies (K48, K63, etc.)
- May require immunoprecipitation for enrichment
- Optimize detection conditions
Other Post-Translational Modifications
SUMOylation
- Include SENP (SUMO protease) inhibitors in lysis buffer
- Use anti-SUMO antibodies (SUMO-1, SUMO-2/3 specific)
- Process samples quickly to prevent deSUMOylation
Glycosylation
- Use glycosylation-specific detection methods
- Consider lectin-based detection
- May require deglycosylation for some applications
Other PTMs
- Hydroxylation, nitration, citrullination require specific protocols
- Use modification-specific antibodies when available
- Optimize sample preparation for each modification type
General Optimization for PTM Detection
Sample Handling
- Process samples quickly to preserve modification status
- Include appropriate inhibitors in lysis buffer
- Keep samples cold during preparation
- Minimize freeze-thaw cycles
- Store samples properly with inhibitors
Antibody Optimization
- Use modification-specific antibodies
- Verify antibody specificity and validation
- Optimize antibody concentration through titration
- Extend incubation times if needed (overnight at 4°C)
- Test with positive and negative controls
Detection Strategy
- Detect modified protein first (more labile)
- Strip membrane and reprobe with total protein antibody
- Normalize modified signal to total protein
- Use sensitive detection methods
- Consider loading more protein if modification is low