Tools to Analyze Protein-Protein Interactions

Conventional methods for studying protein-protein interactions—including co-immunoprecipitation, cross-linking, and pull-down assays—are often labor-intensive and limited in sensitivity. To address these challenges, we asked three leading companies about which state-of-the-art products they are developing to help researchers investigate protein-protein interactions in the lab.

Making SPR easier

Claire Shepherd, Global Product Manager at Cytiva, explains that the company’s Biacore™ SPR systems use surface plasmon resonance (SPR). “SPR was once a technique reserved for specialists,” she says. “However, this technology has evolved over recent decades and has now been widely adopted as a go-to technique for real-time molecular interaction analysis.”

Briefly, SPR for protein-protein interactions involves immobilizing one of the binding partners (called the ligand) on a sensor surface. The second binding partner is then injected over the surface containing the ligand. Finally, binding interactions can be monitored based on changes in the refractive index near the sensor surface.

Shepherd highlights that Biacore SPR is a label-free technique that measures real-time interactions to provide information about affinity, active concentrations, specificity, epitope binding, and complex formation. Furthermore, she stresses that Cytiva’s technology is quite easy to use. “Current Biacore SPR systems provide high-quality data with limited need for training. Users are supported with predefined run and evaluation methods.” In a recent survey, 100% of Biacore™ SPR users reported being satisfied or extremely satisfied with the system’s ease of use.

Biacore systems are fully automated, which reduces hands-on time compared with manual methods such as co-immunoprecipitation and ELISA. Furthermore, there is no molecular weight limit on the interactions that can be studied, which can range from ions and small molecules to whole cells and viruses. Interactions can also be measured from the picomolar to the millimolar range.

Cytiva offers a variety of Biacore SPR systems. For instance, the Biacore X100 is an easy-to-use interaction analysis system that provides data on affinity, kinetics, and active concentrations. Meanwhile, the Biacore 8 series systems are high-throughput and high-sensitivity platforms that deliver high-quality binding data.

Shepherd notes that the costs of analyzing protein-protein interactions with Biacore SPR technology vary depending on the system used and the type of analysis. “However, the flexibility and range of the Biacore portfolio ensure that options are available to suit various budgets and needs.”

Visualizing localized interactions under a microscope

Lawrence Rentoul, Portfolio Lead of ELISA & Specialty Assays at MilliporeSigma (the life science business of Merck KGaA, Darmstadt, Germany, in the United States and Canada), explains that Duolink^® proximity ligation assays (PLA) are ideal for studying endogenous protein-protein Interactions. Such assays provide fluorescent signals for single binding events that can be visualized by microscopy or flow cytometry.

Briefly, this method requires a pair of primary antibodies that bind to the two proteins of interest. Secondary antibodies coupled to short stretches of DNA (called PLA probes) bind to the primary antibodies. When proteins come into close proximity, this DNA undergoes rolling circle amplification and is subsequently bound by a fluorescently labeled probe, generating a signal.

Rentoul notes that Duolink PLA can analyze stable, transient, or weak interactions. “The technique is ideal to detect a single interaction at endogenous levels in cell lines and tissue samples. It can also be considered semi-quantitative for visualizing discrete spots under fluorescence microscopy. Microscopy also allows for an understanding of the localization of protein-protein interactions.”

He also emphasizes that Duolink PLA is versatile. “We see scientists using it in traditional immunohistochemistry setups and automated 96- and 384-well assays. Some researchers use it to confirm and explore protein-protein interactions before potentially deploying other tools.” The ability to use Duolink in both cell lines and native tissue enables both discovery and translational research.

Rentoul stresses that Duolink PLA is straightforward to use. Researchers will need a microscope, two antibodies for the two proteins, and a starter kit (usually at a cost of less than $1,800). “As you scale, you can move away from the starter kits and buy reagents individually in the volumes required, which normally lowers the cost per data point. Duolink is set up to be accessible across a broad range of use cases.”

Measuring interactions without wash steps

Ellen Crummy, Senior Research Scientist at Promega, notes that the company’s Lumit^® Anti-Tag Protein Interaction Reagents are based on its well-known NanoBiT platform, which enables bioluminescence-based detection of protein-protein interactions. In contrast to the company’s live-cell technologies (based on NanoBiT), the Lumit Anti-Tag reagents are designed to detect in vitro interactions.

“Our new Lumit Reagents are very convenient because they allow researchers to measure protein-protein interactions in 30 to 120 minutes without any washing steps,” she notes. The only major requirements for Lumit techniques are two proteins (with two different purification tags) and a luminometer to measure bioluminescence. Promega currently offers Lumit Anti-Tag reagents for five common tags: 6xHis, GST, FLAG (sequence DYKDDDDK), human IgG, and biotin. After the tagged proteins have been expressed, the Lumit reagents can then be used to detect in vitro interactions.

Crummy explains how Lumit Technology is ideal for examining interactions between two proteins with different purification tags. “For example, if Protein A is tagged with 6xHis and Protein B is tagged with GST, you could use Lumit antibodies against 6xHis and GST. One of the antibodies would be conjugated to LgBiT, and the other would be conjugated with SmBiT.”

When the two tagged proteins bind, bringing the antibodies into close proximity, LgBiT and SmBiT interact to form the functional NanoLuc luciferase enzyme. This ultimately generates luminescence that can be measured.

Crummy explains that the Lumit add-and-read protocol is fast and straightforward. She highlights that the technical manual walks users through two simple experiments to optimize the reaction components and concentrations. Researchers can then test their downstream applications by adding small molecule inhibitors or PROTACs. Since Lumit is bioluminescence-based, simple luminometers, which typically do not require calibration, are all that is needed to read the signal.

Overall, Crummy says that Lumit is comparable in cost to technologies like TR-FRET and AlphaLISA. “However, because the technology only requires a simple luminometer, the reader is much cheaper than time-resolved fluorescence-based plate readers.”