Understanding Bio-Layer Interferometry: Principles, Comparison, & Applications 

Biological systems do not exist in an isolated space or a vacuum. Instead, living organisms comprise cells and biomolecules that constantly interact with each other. Enzymes, for instance, catalyze reactions by binding to other proteins or with small molecules and peptides. How these reactions proceed depends on how well the proteins bind to and dissociate from their targets. These processes are known as protein kinetics and dissociation, respectively.

Researchers have developed several lab-based, or in vitro, techniques to study how proteins interact with their targets. Scientists began with enzyme-linked immunosorbent assays (ELISAs), where changes in the concentration of a chromogenic substrate determine how fast an enzyme catalyzes a reaction. Subsequently, researchers developed co-immunoprecipitation, where proteins part of a larger complex are identified by purifying one of those proteins with an antibody. When the need for mass-characterization arose, affinity purification mass-spectrometry (AP-MS) emerged. AP-MS uses a protein as a bait to capture a complete list of biomolecules that interact with the protein.

Although these techniques can help researchers study protein interactions, several caveats reduce their usefulness in generating high-throughput data. With co-immunoprecipitation, researchers can only investigate one protein at a time. Contaminants can also bind to the bait protein in AP-MS, producing vast amounts of useless data. With these shortcomings, scientists required new technologies to assess multiple interactions at a high-throughput rate while minimizing signal noise.

These needs drove scientists to invent a series of technologies that track real-time protein kinetics. The data produced from these technologies facilitate interaction analyses to identify molecules that bind to proteins of interest. Although researchers started with surface plasmon resonance (SPR), they soon built on SPR by developing bio-layer interferometry (BLI). As researchers continue to seek ways to increase the throughput of studying protein-protein interactions, BLI provides a step forward to making protein interaction reliable and robust.

Surface Plasmon Resonance (SPR): The Existing Technology

SPR instruments are the first label-free approach for studying biomolecule interactions. SPRs monitor protein interactions by using a light source. With a light source, SPR begins by reflecting off a gold plate light of a single wavelength. When light strikes the gold plate, its electrons are excited, forming what are called plasmons. The plasmons generate an electric field that indicates how much light is refracted from the plate. 

The protein of interest, in turn, undergoes immobilization on the other side of the gold plate. These protein ligands, along with any biomolecules introduced by a microfluidics system, change the refractive index where the light interacts with the gold plate. During these interactions, a biosensor detects changes to the refractive wavelength. These changes are proportional to the amount of analyte bound to the bait ligand over time. These changes then get recorded as a sensorgram, a line graph that monitors changes in protein interactions with its analytes. 

Through the SPR instrument and the sensorgram, various aspects of protein interactions, including changes in binding affinities, dissociation constants, and molecular interactions, can be monitored.

What Is BLI Technology & How Does It Differ from SPR?

Scientists further developed label-free approaches to track protein interactions with BLI. As such, BLI shares multiple similarities with the technique. Like SPR, BLI is a label-free method, not requiring fluorescent labels to observe proteins and analytes. 

BLI instruments also feature a biomolecule that acts as the bait and other biomolecules that act as the analytes. Finally, all BLIs contain a biosensor that immobilizes a protein of interest and tracks biomolecular interactions.

Despite these similarities, BLIs also differ from SPRs in several ways:

• The nature of the biosensor: Although BLIs employ a biosensor, the type of biosensor differs from SPRs. Instead of a solitary gold plate, BLI biosensors are composed of tips coated with streptavidin, a compound that binds to biotinylated biomolecules for immobilization. The biosensor tips can be transported directly into solutions containing the analytes of interest, known as a dip-and-read format.
• Wavelength interference: Instead of measuring the angle or intensity of light from a light source, BLIs measure the interference pattern of white light. In this setup, white light shining from a light source reflects a bio-layer. The bio-layer is produced when the biosensor tip is immersed in the BLI solution. When the solution contains an analyte, the analyte interacts with the immobilized bait molecule. This, in turn, causes the light to undergo a wavelength shift because the binding event changes the thickness of the bio-layer. It is these shifts that generate the interference patterns for data analysis.
• Use of a solution well: Instead of the analytes moving through a flow system to interact with the biosensor, the analytes remain stationary inside a solution well. This provides a platform for the dip-and-read biosensor to interact with the analytes.

The modifications in the BLI system confer upon it many advantages over SPR for analyzing protein-protein-small molecule interactions:

• High-throughput: BLI’s well system allows hundreds of interactions between baits and analytes to be measured at once. The dip-and-read approach that arises allows protein kinetics to be determined on 96-well or even 384-well plates. This allows analyte interactions to be determined at many different concentrations or hundreds of biomolecular interactions to be assessed at once.
• More cost-effective: Being able to conduct experiments without the need for a flow system reduces the assay’s cost. That’s because the interactions are measured on disposable tips that are dipped into the sample well (as seen in the Octet’s dip-and-read well-based format).
• Flow-free: SPR relies on a flow system to have analytes interact with the bait. However, how undisturbed the flow from the microfluidics system is may adversely affect the interaction data. Contaminants and reagent impurities can hamper the system’s flow, reducing the specificity of the kinetics data.

How To Perform a BLI Experiment

A typical BLI experiment features a series of steps to assay biomolecular interactions:

• Preparing the initial baseline: This is used to determine the baseline wavelength interference before any bait is added. Here, the BLI buffer is added to the system to coat the biosensor and determine the wavelength interference of light without any biomolecules added.
• Loading: The ligand of interest is next added to the biosensor to be immobilized. Any ligands that don’t bind are washed away as the biosensor is coated.
• Measuring the baseline: With the buffer present as a reference layer, the shifts in wavelength interference relative to the initial baseline are determined. This wavelength pattern acts as a reference before any analytes are added.
• Association: This is when the experiment begins. Here, the analytes are introduced into the solution. This action changes the wavelength interference pattern from the white light. This change produces a wavelength shift that the biosensor can measure.
• Dissociation: After the binding kinetics have been sufficiently determined, the BLI buffer is added to wash away the bound analytes. This step helps researchers determine dissociation kinetics, the other half of studying protein kinetics.

The end of the protocol yields a graph that converts changes in wavelength interference into a graph that monitors analyte binding over time. How much and fast the analyte binds and dissociates from the protein of interest depends on the binding rate and how quickly that rate changes.

Applications of BLI

The data generated from a BLI makes it a powerful technique to learn how biomolecules interact with each other. For one, researchers can use BLIs to assess protein kinetics, the study of how proteins associate or disassociate with each other over time. These insights have helped scientists advance biomedical research in several disciplines:

• Studying mitochondrial disease: Researchers have used BLI to study the individual protein sequences that comprise the sole DNA polymerase holoenzyme, DNA polymerase γ. Studying these dynamics may help researchers determine potential factors in how proteins are produced that contribute to mitochondrial diseases.
• SARS-CoV-2 research: More recently, researchers developed and optimized BLI for identifying antibodies that interact with SARS-CoV-2. Most promisingly, the researchers were able to conduct the experiments using 96-well and 384-well plates. With BLI, researchers can complement future efforts to evaluate vaccine candidates and survey antibody responses to prevent and manage any future pandemics.

• Advancing cancer research: Cyclin-dependent kinases (CDKs) act as gatekeepers for the cell cycle by adding phosphate groups to any proteins that bind to the protein. Changes to these binding dynamics may increase the risk of cancer by enhancing the proliferation of cancer cells. By studying these protein dynamics with BLI, researchers can characterize the ways CDKs interact with diverse biomolecules and propel drug discovery efforts in cancer. Researchers can also monitor how CDK inhibitors affect CDK activity with BLIs.

Researchers can also use BLI to investigate interactions between proteins and other types of biomolecules:

• Nucleic acid-protein interactions: The processes that drive life require DNA to be transcribed and translated into mRNA and functional proteins respectively. DNA-binding proteins bind to target DNA sequences and regulate the first of these processes. These proteins can interact with DNA sequences in myriad ways, raising the need to characterize DNA-protein interactions at a high-throughput rate. Established BLI protocols provide binding and dissociation kinetics data to study these interactions further.
• Protein-small molecule binding: Using BLI also helped researchers to study changes in binding dynamics between different small molecules and the 3C-like protease enzyme in SARS-CoV-2.

Bio-Layer Interferometers for R&D

Encouraged by the possibilities that BLI brings to protein characterization and binding research, Excedr has worked with the two companies who kickstarted research in this field—Sartorius and Gator Bio—providing BLI leases to those in need of a creative solution to investing in lab equipment. 

Both Sartorius and Gator Bio have worked tirelessly to produce BLIs that conduct high-throughput protein kinetics research and data analysis. These technologies enabled researchers to produce valuable insights into protein dynamics in the lab and the environment.

• Sartorius: Sartorius boasts the Octet BLI Label-Free Detection System that provides a series of benefits over traditional methods such as ELISAs and SPRs. The machine allows up to 96 samples to be analyzed at once with real-time analysis to assess binding kinetics over time. The Octet also works on crude samples (eg. cell lysates) without contaminants strongly affecting wavelength shifts. Sartorius boasts two additional machines for researchers to conduct BLIs. The Octet R2 to R8s have multiple channels that increase throughput and sensitivity. The other, the Octet RH16 and RH96, includes a robot system to automate sample transfer and reduce operator input in 96-well and 384-well plate formats.
• Gator Bio: Gator Bio hosts the Gator Pro, the primary instrument for conducting BLI experiments. The interferometer has 32 spectrometers to analyze in parallel up to 32 samples. With three sample plates available, up to 1152 samples can be analyzed at once per batch. Gator Bio also hosts many kinds of BLI biosensor probes that bind to several kinds of proteins. These include streptavidin proteins, Strep-Tactin XT proteins, and Custom Anti Strep-tag II probes, each of which are used in different kinds of protein purifications.

Speak with Excedr today

Studying protein interactions has become an essential component of studying the processes that allow us to live. Even well-characterized proteins may still have novel binding interactions yet to be discovered. Each of these dynamics can affect a patient’s health and impact efforts to engineer proteins for societal progress.

Excedr’s leasing program helps you identify the best BLIs to meet your research needs. Once you determine the throughput, experimental applications, and other factors in your decision, we can work with you to determine if our leasing program is right for you. If all goes well, we can create a lease estimate and ultimately help you acquire the best BLI for advancing your protein research. Interested in leasing a BLI? Let us know!