Protein-protein interactions (PPIs) are fundamental to biological processes, and understanding these interactions is crucial for drug discovery, disease research, and molecular biology [1]. A truly effective PPI binding assay should be highly sensitive, quantitative, and capable of assessing interactions in physiologically relevant conditions. Moreover, an ideal assay minimises artifacts, requires minimal sample preparation, and provides robust, reproducible data.
Many existing techniques, such as Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI), and Isothermal Titration Calorimetry (ITC), provide valuable insights but come with inherent challenges. These include the need for surface immobilisation (SPR, BLI), large sample volumes (ITC), or complex labelling requirements (Fluorescence Polarisation, ELISA). These constraints can introduce artifacts, limit throughput, or alter the native binding properties of proteins [2].
To help you navigate these challenges, we provide essential tips to master your binding assay and obtain reliable results. In this article, we explore traditional techniques, their limitations, and key considerations for optimising binding assays to improve reproducibility and accuracy, particularly in the context of in-solution versus surface-bound methodologies.
1. In-Solution vs. Surface-Bound Binding Assays: Choosing the Right Format
A key distinction among PPI assays is whether they take place in solution or require surface immobilisation. This choice significantly impacts the reliability and physiological relevance of results.
- Surface-Bound Assays (e.g., SPR [3], BLI [4], ELISA [5]) require one interaction partner to be immobilized on a sensor chip or well surface. While these methods provide real-time kinetic measurements, surface effects can alter binding properties or introduce steric hindrance, potentially skewing results. Additionally, BLI and ITC require significant quantities of sample, which may limit their applicability for precious or low-yield proteins.
- In-Solution Assays (e.g., ITC [6], and MDS [7]) preserve native protein dynamics by allowing interactions to occur freely in solution. These methods often require minimal sample preparation and avoid artifacts from surface tethering.
Microfluidic Diffusional Sizing (MDS) stands out as an in-solution technique that avoids many of the limitations of surface-bound methods, offering reliable affinity measurements with minimal sample handling. Additionally, MDS requires only 4 µL per size measurement, making it a highly efficient option for low-volume samples.
Figure 1: Common In-Solution vs Surface Bound binding assays [8-12]
2. Protein Expression and Purity: A Critical Factor for Most Techniques
Protein quality directly impacts assay success. For most PPI assays, ensuring properly folded, purified proteins is crucial to obtaining reliable results. Considerations include:
- Expression System Choice: Mammalian, insect, or yeast systems often yield proteins with post-translational modifications essential for native interactions, while bacterial expression may be faster but lack such modifications [13].
- Purification Methods: Techniques like affinity chromatography and size-exclusion chromatography help isolate homogeneous and functional proteins.
- Stability Considerations: Maintaining proteins in optimal buffers, temperatures, and storage conditions minimises degradation and aggregation.
However, not all binding assays require extensive protein purification. MDS, for instance, can analyse interactions directly in complex biological mixtures such as 100% serum, significantly reducing the burden of purification compared to other techniques.
3. Buffer Selection and Optimization
Buffer composition significantly impacts protein stability and interaction strength. Proper buffer selection is essential for maintaining sample integrity and ensuring accurate measurements, but it can also pose challenges for some techniques.
- pH: Matches physiological conditions of the proteins but can influence binding behaviour and stability in some methods.
- Ionic Strength: Can modulate electrostatic interactions but may affect immobilisation efficiency in surface-bound assays like SPR and BLI.
- Detergents: Help stabilize membrane proteins but may interfere with surface-based binding measurements.
- Additives: Glycerol, BSA, or reducing agents (DTT, β-mercaptoethanol) can improve protein stability but must be carefully optimised to avoid unwanted interactions.
MDS is less sensitive to buffer composition variations compared to surface-bound methods, making it particularly well-suited for analysing complex biological samples.
4. Gaining a Full Picture of Protein Interactions
A comprehensive understanding of protein interactions requires selecting assays that provide different types of data. While KD is a valuable parameter, it does not fully describe the nature of binding. Complementary techniques that measure multiple factors can offer a more complete picture of the interaction:
- Stoichiometry: Helps determine the binding ratio between interacting molecules.
- Concentration: Ensures accurate quantification of binding partners.
- Molecular Size: Provides insight into conformational changes and complex formation.
By integrating these different approaches, researchers can build a more complete understanding of protein interactions. MDS can provide a unique advantage by using molecular size to determine stoichiometry and concentration in complex biological samples without immobilisation or purification. This makes it an invaluable tool for studying protein interactions under physiologically relevant conditions, complementing other techniques and enhancing the accuracy of PPI characterisation.
5. Minimising Non-Specific Binding and Background Noise
Non-specific interactions can lead to false positives and poor signal-to-noise ratios. To minimise these:
- Block non-specific sites with BSA or casein.
- Use appropriate controls, such as non-binding protein variants.
- Optimise washing steps to remove unbound proteins.
- Choose high-affinity antibodies or detection reagents in labelled assays.
Because MDS operates in solution and does not require immobilisation, it inherently reduces background noise and non-specific binding artifacts.
6. Reproducibility and Data Analysis
Reproducibility is key to drawing meaningful conclusions. Ensure that:
- Experiments are performed in triplicates or more.
- Independent validation methods confirm interactions.
- Proper statistical analysis is applied to evaluate significance.
- Data visualisation tools are used for clear representation of binding curves and kinetics.
Conclusion
While no single binding assay can address every aspect of protein-protein interaction analysis, choosing the right method is critical. The fundamental distinction between in-solution and surface-bound assays plays a crucial role in data interpretation, experimental design, and overall reliability.
Microfluidic Diffusional Sizing (MDS) emerges as a compelling in-solution alternative, offering an equilibrium measurement that overcomes many of the challenges faced by traditional surface-bound techniques. By enabling the study of native interactions with minimal sample preparation and fluorescent labelling, without requiring surface immobilisation, MDS serves as an essential orthogonal approach to complement existing kinetic assays. Additionally, MDS is particularly well-suited for analysing difficult sample types, such as 100% serum and membrane proteins, where surface-based methods may struggle. While it does not provide kinetic rate constants, its highly accurate affinity measurements make it an invaluable tool for PPI characterisation.
Whether as a primary method or in combination with kinetic assays, MDS enhances the reliability and scope of PPI studies, delivering meaningful insights with fewer experimental constraints.
References
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