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  • April Wong

Surface Plasmon Resonance for Protein-Protein Interactions

Updated: Nov 5


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 (Fig. 1). The change in angle or wavelength (depending on the mode of interrogation) of the reflected light is captured and plotted vs. time to generate a sensorgram.

Figure 1. General setup of a protein-protein interaction on SPR sensor chips. The SPR response is collected in real-time from the light being reflected from the gold film as ligands bind to the capture molecules.

Sensorgrams are used to extract affinity and kinetic data. They can also reveal any specificity and concentration information through the magnitude of the SPR signal. In general, a sensorgram has five phases (Fig. 2). One of the phases is called the association phase, where the 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 between the molecules, causing the signal to decrease. For more details about sensorgrams, please see this blog. One point to note is that affinity and kinetic data are realistically collected under separate experimental conditions as it is challenging to obtain the required sensorgram features to generate both types of data (please read Technote on Manual Injection vs. Pump).

Figure 2. An idealized SPR sensorgram. Phase 2 is the association phase and phase 4 is the dissociation phase. Equilibrium dissociation constant (KD) as well as association and dissociation rates (kon and koff) can be extracted from various phases of a sensorgram. Black ovals represent ligand proteins and the blue V-shaped receptors are capture proteins.

One of the practical applications of SPR is to examine protein-protein interactions. They regulate many cellular processes such as cell cycles, gene expressions, as well as lipid and carbohydrates metabolism. Therefore, a complete understanding of these protein-protein interactions is essential to comprehend the function of cells and other cellular processes necessary for human health. Multiple tools are available for studying these protein-protein 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 (please read Sensors and Surface Chemistry Technote for more details on how molecules can be immobilized on Affinité's sensor chip surfaces). 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. The change in SPR response due to the change in refractive index depends on the amount of mass bound to the capture protein on the SPR sensor chip. 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.

Case Examples of Protein-Protein Interactions

Examples of how SPR can be used to study protein-protein interactions can be found on our Research Applications page (please see appnotes on Detection of Hemagglutinin and Protein-Protein Interaction Screening for more details on how molecules can be immobilized on Affinité's sensor chip surfaces). In the first example, a calibration curve was obtained to demonstrate the feasibility of Affinité P4SPR™ to quantify viruses in vaccine lots by detecting hemagglutinin (HA). Anti-HA antibodies were immobilized on the sensor chip to detect HA. One can observe the increasingly larger association curve as each increasing HA concentration was injected. In another example, the protein EntF, known to play a role in the biosynthesis of enterobactin in E. coli, was immobilized and introduced to either EntA, EntB, EntC, or EntD protein from the same pathway to search for interacting binding partners. Association curves were observed for some proteins but not for others. This indicates that only some of the tested protein molecules interacted with the immobilized EntF.

Advantages of Using SPR

In a nutshell, surface plasmon resonance for protein-protein interaction is the most commonly used method to study molecular interactions in real-time. Some of the key benefits of using SPR are discussed below:

1. Observation of binding interactions in real-time. One of the most important benefits of using SPR for understanding protein-protein interactions is observing the results in real-time as a sensorgram, unlike other endpoint-based assays such as immunoassays. A sensorgram provides information about affinity and kinetic information as well as concentration and specificity.

2. 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 until a time requirement is met or the ligand protein dissociates, for example. Incubation times for SPR experiments are shorter as well.

3. No labelling is required. SPR response relies on the change in local surface refractive index. Therefore, it does not rely on detecting traditional labels such as fluorophores, or an enzyme-linked antibody to produce a coloured product. The addition of labels and enzymes may mask the true protein-protein binding interaction between the capture and ligand proteins.

4. Reusable sensor chip. Affinité's SPR sensor chips are reusable in certain cases. For example, calibration curves can be performed on the same chip in the same experimental run by injecting the lowest to the highest ligand concentrations, as seen in the Detection of Hemagglutinin appnote. One can use the sensor multiple times, which makes protein binding SPR more economical and affordable. In one recent publication by Djaileb et al., the sensor chip could be regenerated 9 times even after serum samples were used [1].


Surface plasmon resonance is a very simple and rapid strategy that can examine protein-protein interactions without having to use labels. Sensorgrams that are collected from an SPR experiment in real-time contain valuable information pertaining to specificity, concentration, affinity, and/or kinetics.

The Affinité Advantage

Affinité Instruments’ P4SPR™ is a very user-friendly, compact, and portable instrument. In addition, samples do not need much preparation and can be manually injected into the instrument. The P4SPR™, compared to a traditional immunoassay such as ELISA, provides fast, real-time affinity and/or kinetic data.

Simplicity - Fast training, fast results

Versatility - Pharmaceutical, biosensing, assay development applications

Economy - Affordable, accessible

We help life science labs and biotech companies to do rapid assay development and characterization. Feel free to reach out to us about the expertise we offer at


[1] Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals - Analyst (RSC Publishing)