Go All-In with the Top Methods for Label-Free Protein Characterization
Doing research is a gamble with much at stake. Out of infinite combinations and molecular structures, you might discover the next blockbuster drug, or waste billions betting on the wrong molecule. To stack the odds in your favor, you need high value cards in your hand. For drug developers, this means choosing tools that help to identify leads with the best chance of success. In this blog, we talk about two well established label-free techniques for biomolecular interaction analysis—Bio-Layer Interferometry (BLI) and Surface Plasmon Resonance (SPR). Learn how these real-time techniques can empower research and biopharma analysis workflows when used individually, or together.
This article is posted on our Science Snippets Blog
Getting an edge in protein analysis
Understanding the binding kinetics between molecules or simply measuring the concentration of active analytes are fundamental characterizations in both basic research and drug discovery. For example, when developing a monoclonal antibody biologic, it’s important to know if it binds to the clinical target. If so, how tight is the interaction? And what are the rates of association and dissociation? These attributes are directly related to how well the drug will eventually perform and are used to optimize and validate lead candidates.
Unlike labeled end-point methods like ELISA, label-free real-time methods for detecting biomolecular interactions don’t require labeling of any of the interactants. Because of that, you can design simpler assays. The lack of fluorescent labels and/or secondary detection reagents also eliminates interference or related experimental artifacts, leaving you with clean data that better captures biologically relevant interactions.
Building a winning hand with label-free analysis
The two most popular methods for label-free detection are BLI and SPR. While these technologies are quite powerful individually, together they’re a winning combo in terms of answering the widest range of questions. Sartorius is the only provider of both industry-leading technologies under one trusted brand. The fluidic-free Octet® BLI range is synonymous with speed and ease-of-use, while the Octet® SPR range has patented injection tools that really simplify multi-concentration studies.
How does label-free detection work?
BLI and SPR use optic-based biosensors that convert a binding event into a biophysical signal that can be plotted as a response curve, or sensorgram. In either assay, one binding partner (ligand) is immobilized on the biosensor surface, while the other (analyte) remains in solution.
You can monitor analyte-ligand complex formation in real time as distinct phases on the sensorgram. The baseline phase shows the response of the immobilized ligand to the assay buffer alone. When the analyte is introduced, binding to the ligand is recorded in the association phase. Switching back to assay buffer starts the dissociation phase, where the analyte naturally dissociates from the ligand. This data is then used to calculate kinetic constants, like KD, ka, and kd.
Figure description: The Octet® BLI and SPR systems use a dip-and-read and continuous flow system, respectively. The distinct phases in a typical sensorgram are baseline, association, dissociation, and regeneration back to baseline.
What is bio-layer interferometry?
BLI technology uses fluidic-free dip-and-read biosensors, which is where the ligand is immobilized. The biosensor moves to a 96-or 384-well plate and is “dipped” into the sample—this is a robust and simple way to introduce the analyte to the sensor surface. When analyte binds to the immobilized ligand, BLI can sense the change in thickness of the biolayer on the sensor tip by measuring the change in interference pattern between light waves.
What is surface plasmon resonance?
Like BLI, SPR is an optical, label-free, real-time technology for measuring molecular interactions. SPR uses a continuous flow system to deliver analyte to the immobilized ligand on a biosensor chip. This technique measures the shift in the angle of total reflected light on the sensor surface. Binding of analyte to ligand causes a change in the refractive index on the sensor surface resulting in the shift seen on the sensorgram.
Choosing the best label-free technique
Both BLI and SPR are widely published with their own unique advantages, so the right choice depends on the application and the size range of molecules involved. SPR, for example, is a more sensitive technique for measuring interactions with small molecules and fragments. BLI on the other hand, is better suited for pharma-level high throughput analysis, with the added flexibility to perform assays in crude lysates during process optimization or bioprocess monitoring.
When used orthogonally, BLI and SPR are powerful, complementary tools for the characterization of biomolecules, such as antibodies, fragments or other biotherapeutic proteins in research and biopharma analysis workflows.
To explore more on this topic, read A Compendium for Successful BLI and SPR Assays. It includes detailed chapters about assay design and focused discussions on avidity, surface regeneration and other expert advice on getting the best data. Also, Sartorius is currently running a one-of-a-kind bundle promotion for labs interested in both Octet® BLI and SPR instruments.
