Last Updated on
September 27, 2023
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.
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.
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 modifications in the BLI system confer upon it many advantages over SPR for analyzing protein-protein-small molecule interactions:
A typical BLI experiment features a series of steps to assay biomolecular interactions:
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.
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:
Researchers can also use BLI to investigate interactions between proteins and other types of biomolecules:
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.
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!