Biomolecular binding affinity
Biomolecular binding affinity refers to the strength of the interaction between two or more biomolecules, such as proteins, nucleic acids, or small molecules. It quantifies how likely and how tightly these molecules will bind to each other. The binding affinity is a measure of the attraction or affinity between the molecules and is typically expressed as a dissociation constant (Kd) or equilibrium constant (K).
A high binding affinity indicates a strong interaction between the molecules, where they are more likely to bind and remain bound together. Conversely, a low binding affinity suggests a weaker interaction, and the molecules are less likely to form stable complexes.
The binding affinity is influenced by various factors, including the chemical and physical properties of the interacting molecules, the shape and complementarity of their binding sites, and the surrounding environmental conditions such as temperature and pH. It plays a crucial role in many biological processes, such as enzyme-substrate interactions, receptor-ligand interactions, protein-protein interactions, and drug-target interactions.
Determining and understanding the binding affinity between biomolecules is essential in fields such as drug discovery, structural biology, and molecular biology. It helps researchers design and optimize drug candidates, study protein-ligand interactions, and gain insights into the mechanisms underlying biological processes.
Measurement of binding affinity:
There are several methods used to measure biomolecular binding affinity, and the choice of method depends on the nature of the molecules being studied and the desired level of detail. Here are a few commonly employed techniques:
- Surface Plasmon Resonance (SPR): SPR measures changes in refractive index near the surface of a sensor chip to monitor binding interactions. One molecule is immobilized on the chip, while the other is flowed over it. The binding event results in a shift in the refractive index, which is used to determine the binding affinity. .
- Isothermal Titration Calorimetry (ITC): ITC measures the heat released or absorbed during a binding event. One molecule is placed in a sample cell, and the other is titrated into it. The heat generated or absorbed is measured, allowing the determination of the binding affinity, stoichiometry, and thermodynamic parameters.
- Fluorescence-based Assays: Fluorescent techniques exploit changes in fluorescence properties upon binding. For example, fluorescence resonance energy transfer (FRET) measures the transfer of energy between two fluorophores, one attached to each molecule. Changes in FRET efficiency can be used to determine binding affinity.
- Electrophoretic Mobility Shift Assay (EMSA): EMSA measures the migration of biomolecules through a gel matrix in response to an electric field. A shift in mobility occurs when a complex is formed between two interacting molecules. The degree of shift can be used to assess binding affinity.
- Biosensor-based Assays: Various biosensor platforms, such as biolayer interferometry (BLI) and quartz crystal microbalance (QCM), can be employed to measure binding affinity. These methods monitor changes in mass or surface properties in response to binding events.
- Microscale Thermophoresis (MST): MST utilizes changes in the movement of molecules in a temperature gradient to quantify binding affinity. It measures changes in fluorescence intensity or thermophoretic movement of fluorescent labeled molecules as they interact.
- The iRiS Kinetics technology presented here, is a significant improvement over SPR and BLI.
These are just a few examples of the techniques historically available for measuring biomolecular binding affinity. Each method has its advantages and limitations, and the choice of method depends on factors such as the molecules involved, the required sensitivity, and the experimental conditions.
Label-Free Interaction Analysis
With the rapidly expanding focus on monoclonal antibody products and other biologic therapeutics in pharma and biotech, reliable and efficient Biomolecular binding affinity analysis has established itself as an essential research tool. Biomolecular kinetic binding data has proven invaluable for everything from drug delivery to immunoassay development.
How to measure binding affinity
Previous biomolecular binding assays used technologies that often relied on highly specific probes and antibodies and were purpose-built to study a single interaction. Although precise, the laborious nature of these techniques grinds research to a halt — a death knell in the fast-paced modern research landscape. As a result, novel label-free interaction analyses have emerged that take advantage of the physical properties of nanoscale biomolecular interactions.
Advances in how to measure peptide, protein, small molecule, and antibody binding affinities have resulted in several new analytical modalities, such as surface plasmon resonance (SPR) affinity measurements, calorimetric analysis, or quartz crystal microbalance (QCM). Thanks to the emergence of these techniques, researchers have moved beyond classic techniques like equilibrium dialysis measurement of antibody affinity.
At axiVEND, we specialize in interferometric reflectance imaging sensor (iRiS) analysis. Silicon chips are layered with SiO2 embedded with probes of your choice. As samples are added, substrates bind to the embedded probes, accumulating biomass and changing the thickness of the optical layer. This added biomass alters the optical qualities of the media, causing a phase shift in reflected light. These changes are visualized and quantified in real time, providing in-depth kinetics data for hundreds of probes without complicated multi-step procedures and costly, perishable reagents.
Comprehensive: Whether your research group relies on protein binding analysis, peptide binding tests, small molecule binding analysis, antibody affinity measurements, or all of the above, the iRiS Kinetics MX-100 has you covered.
Highly multiplexed: Painstakingly screening one probe at a time can drag research and innovation to a halt. With our highly multiplexed assay, you can test using 100s of probes in only 2 hours, allowing you to generate what would once be weeks’ worth of data in the time it takes to run a standard PCR.
Quantitative: Whether you’re screening biomolecule binding to cofactors, inhibitors, or novel small molecule substrates, the MX-100 produces publication-worthy data with each run. Moreover, with its fully-automated operational design, user errors are minimized, allowing laboratory personnel of any experience level to generate data with the utmost confidence.
Repeatable: With industry-leading precision and streamlined operating procedures, you can easily replicate experiments with the iRiS MX-100. Why waste weeks on a false lead when you can rerun your experiment to confirm your results on the same day?
Convenient: The iRiS MX-100 is a compact, low-cost, fully-automated protein binding instrument with inexpensive consumables. Why wait for data from off-site facilities when you can bring precision results to your benchtop with room to spare?
Highly sensitive: Capturing small molecule binding affinity can be challenging with some analyzers — not the case with the MX-100. Our device performs kinetic binding measurements on compounds with a molecular weight of <250 Da weight. In addition, protein, nucleic acid, antibody, and small molecule binding analysis can be performed using samples as small as 100 microliters — don’t let limited sample sizes hamper the scope of your investigations again.
