Protein-protein interactions are at the heart of most biological processes. SPR provides the kinetic and affinity data needed to characterize these interactions with precision.
Introduction
Surface plasmon resonance (SPR) is one of the most commonly used methods to study the interaction of biomolecules in a complex environment in real-time. In this process, the incident light interacts with surface plasmons on a thin gold film to which ligands are attached. The change in angle or wavelength of the reflected light is captured and plotted vs. time to generate a sensorgram.
Sensorgrams are used to extract affinity and kinetic data. They can also reveal specificity and concentration information through the magnitude of the SPR signal. In general, a sensorgram has five phases. One phase is the association phase, where binding interaction between the ligand and capture molecule begins and stabilizes when equilibrium is reached. In the dissociation phase, a wash buffer is introduced to dissociate the interaction, causing the signal to decrease.
Protein-protein interactions regulate many cellular processes such as cell cycles, gene expressions, as well as lipid and carbohydrate metabolism. Multiple tools are available for studying these interactions. However, SPR is the best tool to observe and understand these interactions in real-time without labeling the molecules.
SPR Assay Setup
Setting up an SPR experiment for examining protein-protein interactions involves attaching a capture protein molecule onto the SPR sensor chip surface. Then, the protein ligand of interest is injected into a microfluidic channel, which is in contact with the sensor chip. As the ligand flows into the channel, it starts binding to the capture protein, causing the refractive index to change. Depending on the experimental setup, the resultant SPR sensorgram is used to extract information about the specificity, concentration, affinity, and/or association and dissociation rate of the protein-protein interaction.
Advantages of Using SPR
Real-time observation. One of the most important benefits is observing the results in real-time as a sensorgram, unlike endpoint-based assays such as immunoassays. A sensorgram provides information about affinity and kinetic information as well as concentration and specificity.
Less laborious and time consuming. Unlike immunoassays such as ELISA, SPR is less laborious and time consuming. There is no need to wash wells by pipetting as the wash buffer can simply be injected. Incubation times for SPR experiments are shorter as well.
No labelling required. SPR response relies on the change in local surface refractive index. It does not rely on detecting traditional labels such as fluorophores, or an enzyme-linked antibody. The addition of labels and enzymes may mask the true protein-protein binding interaction.
Reusable sensor chips. Affinité's SPR sensor chips are reusable in certain cases. A regeneration buffer can be used to dissociate the interaction between the ligand and analyte, and the sensors can be reused, making SPR more economical and affordable.
Conclusions
Surface plasmon resonance is a very simple and rapid strategy that can examine protein-protein interactions without having to use labels. Sensorgrams collected from an SPR experiment in real-time contain valuable information pertaining to specificity, concentration, affinity, and/or kinetics.