When answering research questions related to biomolecular interactions (eg. affinity, on rates, off rates, etc.) in the fields of molecular biology or chemistry, scientists have a number of experimental techniques from which to choose. Radioligand binding and fluorescence labeling are two techniques that have traditionally been employed to study molecular affinity. Now, however, researchers are increasingly turning to methods such as surface plasmon resonance (SPR) to quantify properties of biomolecular interactions.
Radioligand Binding and Fluorescent Labeling Research both require labeling.
To perform radioligand binding, a molecule must be labeled with a radioactive biochemical substance. As the ligand is displaced from the binding site, radioactive decay increases. This decay is measured using scintillography, providing an estimate of ligand binding.
To employ fluorescence binding, this methodology requires the ligand to be labeled with a fluorescent molecule so that scientists can measure the depolarization of emitted light (as with fluorescence polarization) or energy transfer (as with fluorescence resonance energy transfer, or FRET).
Perhaps the biggest downside of these two techniques is that the molecule must be labeled. The labeled construct may differ slightly in its molecular properties, which could introduce error into the data. Additionally, radioligand binding and fluorescent labeling have a lower sensitivity and specificity than newer methodologies.
Surface Plasmon Resonance is a newer technique with Widespread Application.
Surface plasmon resonance (SPR) is a relatively new technology (the first commercial instrument was introduced in 1990). As molecules couple to a gold sensor chip, they are excited with polarized light. The change in mass on the surface results in changes in the local refractive index near the gold layer which can be very accurately measured. This has some advantages over traditional technologies, including:
- No labeling is required.
- SPR can be used to determine ligand affinity with extreme precision.
- It can be used to accurately determine binding constants of molecules.
- SPR has very high sensitivity and specificity
- SPR is quantitative. It can be used to characterize kinetics, concentration, adsorption, affinity, and adhesion of molecules.
- It permits real-time monitoring of biomolecular interactionsResults obtained are highly accuracy and reproducible..
As more is learned about the unique advantages of SPR, it is increasingly adopted by researchers seeking high quality methodologies to answer important questions in molecular biology or chemistry. Reichert SPR technologies have exquisite sensitivity and reproducibility of measurements. With scientific journals increasingly pushing for inclusion of quantitative methods for manuscript publication, SPR represents an excellent method for producing high quality, publishable research.