Over the past few years, many techniques have been developed that have enabled the quantification of biomolecular interactions, providing binding affinities, kinetics and/or thermodynamics of the interactions. This has allowed for in depth characterization of biological interactions. Traditionally, binding affinity has been used to determine how strong the interaction is between two biomolecules; however, the binding affinity alone doesn’t tell the whole story. As we mentioned in a previous blog post, affinity is really only the tip of the iceberg when it comes to understanding the nature of an interaction. When paired with kinetics, binding affinity helps us gain a fuller understanding of the biomolecular interactions under study, which is extremely valuable in applications such as drug discovery and understanding molecular disease mechanisms.
Blog Overview
- Which biomolecular interaction technique best suits my research?
- Microscale Thermophoresis (MST)
- Isothermal Titration Calorimetry (ITC)
- Biolayer Interferometry (BLI)
- Surface Plasmon Resonance (SPR)
- Localized Surface Plasmon Resonance (LSPR)
- Conclusion
Which biomolecular interaction technique best suits my research?
Currently, the four most commonly used techniques for biomolecular interactions are Microscale Thermophoresis (MST), Isothermal Titration Calorimetry (ITC), Biolayer Interferometry (BLI), Surface Plasmon Resonance (SPR), and Localized SPR (LSPR). Each technique has its advantages and disadvantages. Read the MST, ITC BLI, SPR & LSPR comparisons below to determine the optimal technique for your research.
See Cheat Sheet Here
Microscale Thermophoresis (MST)
Microscale Thermophoresis (MST) is a technique that measures the motion of fluorescent molecules along microscopic temperature gradients. A molecule’s thermophoretic property, or movement along a temperature gradient, is determined by the molecule’s size, charge and hydration shell. When the molecule binds to an interacting partner, one or more of these parameters may change, resulting in a change in its thermophoretic movement. Measuring these changes can then allow for quantification of the affinity of the interacting partners.
Advantages:
- Small sample size
- No sample immobilization
- Low sample consumption
- Ability to measure complex mixtures (i.e. cell lysates, serum, detergents, liposomes)
- Wide size range of interactants (ions to MDa complexes)
Disadvantages:
- Requires internal fluorescence or fluorescent labelling (affected by labeling efficiency, specificity and fluorescence quenching)
- No kinetic information (i.e. association and dissociation rates)
- No concentration analysis
- Potential for conflicting, complementary or confounding influence on all three parameters (molecular size, charge and hydration shell), which can affect data interpretation
Isothermal Titration Calorimetry (ITC)
Isothermal Titration Calorimetry (ITC) is a technique used for quantitative thermodynamic characterization of a wide variety of biomolecular interactions. The fundamental components of ITC are a reference cell filled with solvent, a sample cell, and an injection syringe for ligand titration. ITC measures in real time the heat evolved during molecular interactions when a sample in the syringe is titrated into another in the sample cell. Data is collected by measuring the power needed to maintain a constant temperature between the sample cell and the reference cell. This normalizing power is then measured as a function of time and is used to determine the heat evolved on association of a ligand with its binding partner.
Advantages:
- Ability to determine multiple thermodynamic binding parameters (i.e. stoichiometry, association constant, and binding enthalpy) in a single experiment
- Requires no modification of binding partners
- Automated ITC instruments now available
- New calorimeters require smaller sample volume (Note, smaller volume results in smaller heat signal, which can affect signal to noise ratio).
Disadvantages:
- Large sample quantity needed
- Some platforms are limited to affinity determination and will not measure binding kinetics (association and dissociation rate constants)
- Non-covalent complexes may exhibit small binding enthalpies as signal is proportional to the binding enthalpy
- Slow with a low throughput (0.25 – 2 h/assay), not suitable for HTS
Biolayer Interferometry (BLI)
Bio-Layer Interferometry (BLI) is an optical analytical technique used to quantify biomolecular interactions. It analyzes the interference pattern of white light reflected from two surfaces on a fiber optic biosensor tip – a layer of immobilized protein on the fiber optic sensor tip, and an internal reference layer. The binding of molecules in solution to the biosensor tip causes a shift in the interference pattern. This shift is monitored in real time to identify, quantify and characterize proteins and other biomolecules in solution.
Advantages:
- Label-free detection
- Real-time data
- No reference channel required
- Crude sample compatibility
- Fluidic-free system so less maintenance needed
Disadvantages:
- Immobilization of ligand to surface of tip required
- Low sensitivity (100-fold lower sensitivity of detection compared to SPR)
Surface Plasmon Resonance (SPR)
Surface plasmon resonance (SPR) is a label-free technique that allows researchers to quantitatively analyze binding between biomolecules. It typically consists of a sensor made up of a thin film of gold, and an incident light that collectively excites electrons of a conduction band in the gold film and creates a coherent plasmon oscillation, or resonance. Processes which alter the local refractive index such as adsorption of biomolecules onto the gold sensor layer can therefore be monitored in a surface sensitive fashion by recording the shift in resonance. The shift in resonance can then be used to determine the kinetic constants kon, koff and KD.
Advantages:
- Label-free detection
- Real-time data acquisition (i.e. quantitative binding affinities, kinetics and thermodynamics)
- High throughput capabilities
- Very sensitive with reproducible results
- Measures a wide range of on rates, off rates and affinities
- Relatively small sample consumption
Disadvantages:
- High cost of ownership
- Ongoing fluidic maintenance
- Steep learning curve
- Specialized technicians or senior researchers required to run experiments
- Immobilization of one of the binding partners required
Localized Surface Plasmon Resonance (LSPR)
Localized surface plasmon resonance (LSPR) has been studied for many years as an accessible and affordable alternative to SPR, and is the main technique used in Nicoya’s SPR instruments. LSPR is generated by metal nanoparticles, typically gold or silver, instead of the continuous thin film of gold used in traditional SPR. LSPR produces a strong resonance absorbance peak in the visible light spectrum, which is very sensitive to the local refractive index around the nanoparticle. LSPR therefore measures changes in the position of the absorbance maxima, rather than the angle of reflectance as in traditional SPR.
Advantages:
- Affordable instrument and sensors
- Portable and suitable for small benchtops
- High throughput capabilities
- Simplified operation and maintenance
- Robust against vibration and noise
- Less interference from buffer mismatch and temperature drift
Disadvantages:
- Immobilization of one of the binding partners required
Conclusion
Now that you’ve learned about the advantages and disadvantages of MST, ITC, BLI, SPR and LSPR, you can decide which interaction technique is the best fit for your research. Given the advantages and disadvantages of these five techniques, SPR is recognized as the most versatile technique, and is therefore widely considered the gold standard. However, as we covered earlier, the complexity and cost associated with this technique has been the chief hindrance factor in its widespread adoption. Fortunately, Nicoya’s OpenSPR uses LSPR technology to make the technique more accessible and affordable. OpenSPR provides researchers with these main benefits:
- Benchtop: compact and very cost effective, which helps researchers avoid costly & inconvenient core facilities.
- Real-time data: helping researchers publish faster with label-free binding kinetics & affinity data.
- User-friendliness: ability to easily train anyone in the lab to become an SPR expert with our user-friendly solution.
- Low maintenance: not requiring expensive service contracts but easily fixable by you so you can focus on your research.
