Measuring molecular interactions in solution or on a surface

April 21, 2022

To immobilise or not to immobilise? That is the question

There are several ways to categorise affinity assays. For example, binding can be measured in solution or on an immobilised ligand. And you might be pondering which is the right method for your needs and experimental conditions. The decision on whether measuring binding affinity in solution or on a surface should be weighed against advantages and disadvantages. We have summarised them here for you. 

Not to immobilise

Assays that measure binding in solution might mimic the natural binding conditions. Some require labelling, others do not. However, most of the methods that study binders in solution, such as isothermal titration calorimetry (ITC) and analytical ultracentrifugation (AUC), are low-throughput in nature: only one ligand can be tested at a time. Therefore, they are suitable for screening a small number of biomolecules. Also, they rely on using the same buffer conditions for all measurements.

In solution assays measure absolute affinity values and reveal multiple thermodynamic binding parameters (i.e. stoichiometry, association constant and binding enthalpy) in a single experiment. However, they are not capable of providing information on the kinetics of the interaction - the rate of complex dissolution, koff, and the rate of complex formation, kon. While measurements in solution allow for the analysis of proteins in their native form, some methods, such as ITC, can require rather large amounts of protein per measurement.

A few methods, such as microscale thermophoresis (MST), allow for high-throughput and low-sample consumption. The main drawback of this method is the fact that it relies on labelling of one of the binding partners: this can alter the protein structure and, consequently, affect the binding interaction. Therefore, it is important to carefully design your experiments and labelling strategy.

To immobilise

Several affinity assays, such as ELISAs, gel-shift assays, pull-down assays and surface plasmon resonance (SPR), rely on ligand immobilisation. If you are working in the context of drug discovery and you need high-throughput, these would most likely be your methods of choice. In most cases, a low concentration of material suffices, analytes do not need to be labelled and the sample can be recovered. These methods facilitate binding kinetics measurements (kon and koff), complex epitope mapping, epitope binning and competition assays. 

A potential limitation of these techniques is that your ligand may not maintain its native conformation upon immobilisation or may be oriented in a way that sterically hinders the binding. You would also need to design your experiments carefully to control non-specific binding effects, spot artefacts and select the most suitable immobilisation technique. 

Tricks of the trade and HexagonFab Bolt

If the immobilisation of your ligand is troublesome, try the other way around: use your ligand as your analyte and vice versa. This might not be possible with all methods though. While in most techniques the signal is maximised by using larger biomolecules as analytes and smaller ones as ligands, HexagonFab Bolt allows you maximum flexibility. Since this tool detects molecules’ charge rather than their mass, you can choose to immobilise any biomolecule without sacrificing the sensitivity of the experiment. 

Ligands with free amino groups, for example in lysine residues, can be immobilised on surfaces bearing epoxide groups. In other cases, anti-ligand antibodies, biotin or other tags on platform or sensor chips can help you orient the immobilised ligands in a certain direction. Our product, the HexagonFab Bolt, takes advantage of streptavidin-coated sensors to immobilise any biotin-decorated ligands you wish to test.   

We are soon introducing a new, user-friendly tool to address your changing analytical requirements: The HexagonFab Bolt. Stay tuned!