MBE Protein assessment
- 表达层面：Western Blotting（WB）和 ELISA 适合检测蛋白质丰度变化或翻译后修饰（PTM）。
- 相互作用层面：Co-IP、Pull-Down 和质谱（MS）用于解析蛋白复合物组成。
- 动态层面：FRET 可实时监测活细胞中蛋白互作的时空动态。
技术互补性示例¶
- WB → Co-IP → MS：
先用 WB 确认蛋白表达水平，再通过 Co-IP 富集复合物，最后用 MS 鉴定互作蛋白。 - Pull-Down → FRET：
体外验证直接互作后，用 FRET 在活细胞中验证动态过程。
1. Experimental Purpose¶
- Scientific Question: Are specific proteins expressed, modified, or forming complexes under particular cellular conditions?
- Design Rationale: Protein analysis techniques enable detection, quantification, and characterization of protein-protein interactions critical for understanding cellular pathways
- Follow-up Studies: Functional validation of protein interactions, structure-function analysis, development of targeted interventions
2. Model System¶
- Primary Systems: Cell lines, tissue lysates, purified protein preparations
- Rationale: These systems provide sufficient protein yield while maintaining native conformations and interactions
- Alternatives:
- In vitro reconstituted systems (pros: defined components; cons: may lack cellular context)
- In vivo animal models (pros: physiological relevance; cons: complex background, ethical considerations)
- Patient samples (pros: clinical relevance; cons: limited availability, heterogeneity)
- Ethical Considerations: Responsible antibody production, ethical sourcing of biological materials, data reproducibility
3. Measurement Approach¶
- Common Elements:
- Optimized protein extraction methods
- Appropriate buffer conditions to maintain native interactions
- Validated antibodies or detection reagents
- Inclusion of appropriate controls
- Technical Replicates: Minimum triplicate measurements for quantitative assessments
- Potential Biases:
- Antibody cross-reactivity (validate with knockouts/knockdowns)
- Extraction efficiency variations (standardize protocols)
- Artificial interactions during lysis (crosslinking strategies)
- Overexpression artifacts (use endogenous levels when possible)
4. Group Setting¶
- Experimental Groups:
- Test: Samples from treatment conditions of interest
- Control 1: Untreated/baseline samples
- Control 2: Negative controls (non-specific antibodies, irrelevant proteins)
- Control 3: Positive controls (known interactors or expression levels)
- Controlled Variables: Protein concentration, lysis conditions, incubation times, detection parameters
- Biological Replicates: Minimum 3-5 independent experiments for statistical validity
- Modified Design: Include time-course analysis, dose-response relationships, or genetic perturbations
5. Data Analysis & Presentation¶
- Common Analysis Elements:
- Normalization to loading controls or reference proteins
- Statistical comparison between experimental conditions
- Quantification of band intensities or signal strength
- Correlation with functional outcomes
- Presentation Approaches:
- Representative blot/gel images with molecular weight markers
- Quantitative graphs with error bars
- Network diagrams for interaction studies
- Co-localization images for spatial information
6. Technique Comparison¶
| Feature | Western Blotting | Co-IP | Pull-Down Assay | Mass Spectrometry | FRET | ELISA |
|---|---|---|---|---|---|---|
| Primary Use | Protein detection and semi-quantification | Endogenous protein-protein interactions | Direct protein-protein binding | Comprehensive protein identification and quantification | Real-time protein interactions in living cells | Sensitive protein quantification |
| Sensitivity | Moderate (nanogram range) | Moderate (depends on antibody) | Moderate (depends on bait affinity) | High (picogram range) | Moderate (requires sufficient fluorophore expression) | Very high (picogram to femtogram range) |
| Specificity | Good, antibody-dependent | Good, antibody-dependent | Good, depends on bait purity | Very good with proper controls | Good for direct interactions (<10nm) | Excellent (sandwich format) |
| Quantification | Semi-quantitative | Qualitative to semi-quantitative | Qualitative to semi-quantitative | Highly quantitative with proper controls | Quantitative for interaction dynamics | Highly quantitative |
| Throughput | Low to moderate | Low | Low | High | Low to moderate | High |
| Time Required | 1-2 days | 1-2 days | 1-2 days | 2-3 days (including analysis) | Hours to days (construct preparation) | Hours |
| Cost | Moderate | Moderate | Moderate | High | High (equipment) | Moderate |
| Equipment | Gel apparatus, transfer system, imaging | Basic lab equipment | Basic lab equipment | Mass spectrometer, LC system | Specialized fluorescence microscope | Plate reader |
| Technical Expertise | Moderate | Moderate | Moderate | Advanced | Advanced | Basic to moderate |
| Best For | • Protein expression levels • Post-translational modifications • Confirming specific proteins • Relative comparison between conditions |
• Endogenous protein complexes • Verification of interactions • Identifying components of complexes • Native interaction conditions |
• Direct binding partners • Testing specific interactions • In vitro confirmation • Domain mapping |
• Unbiased interaction discovery • Comprehensive proteome analysis • PTM identification • Complex composition |
• Real-time interaction kinetics • Spatial localization of interactions • Conformational changes • Living cell studies |
• Precise protein quantification • Biomarker detection • High-throughput screening • Clinical samples |
| Limitations | • Limited quantification • Antibody availability • Single protein focus • Size limitations |
• Non-specific binding • Transient interactions may be missed • Requires good antibodies • Indirect interactions indistinguishable |
• Artificial conditions • Overexpression artifacts • May miss weak interactions • Limited to binary interactions |
• Expensive equipment • Complex data analysis • Sample preparation critical • Low abundance proteins challenging |
• Requires protein tagging • Background fluorescence • Limited to close interactions • Complex setup |
• Limited to soluble proteins • Antibody pair requirement • Limited structural information • No interaction details |
7. Complementary Usage Strategy¶
- Expression Analysis: Begin with Western blotting to confirm protein expression and basic modifications
- Interaction Screening: Use Co-IP or pull-down assays to identify potential interacting partners
- Comprehensive Mapping: Apply mass spectrometry for unbiased identification of complex components
- Dynamic Analysis: Employ FRET to study real-time interaction kinetics in living cells
- Quantitative Assessment: Utilize ELISA for precise quantification of specific proteins
- Integrated Approach: Combine techniques for robust validation:
- Western blotting to confirm protein expression
- Co-IP to identify native protein complexes
- Pull-down to verify direct interactions
- Mass spectrometry to comprehensively map interaction networks
- FRET to validate interactions in living cells
- ELISA to precisely quantify changes in protein abundance
This multi-technique strategy leverages the strengths of each method while compensating for individual limitations, providing a comprehensive understanding of protein interactions that is critical for elucidating cellular pathways and disease mechanisms.