Understanding how proteins interact is fundamental to biologics development. Biolayer interferometry (BLI) makes these invisible interactions visible with real-time, precise visualization of binding interactions.

What is BLI?

Biolayer interferometry (BLI) is an optical, label-free technique that measures real-time binding at a sensor surface. BLI captures binding events as they happen, so it can measure both the rates and extent of molecular interactions, giving it broad appeal for quantitative kinetic and affinity characterization throughout biologics discovery and development.

BLI works by analyzing interference patterns created when white light passes through specialized biosensors. Here’s how the technology works:

The instrument shines white light through a fiber optic cable and into a glass biosensor. The biosensor tip is functionalized with capture molecules that bind target proteins of interest when placed into solution. As proteins bind and the layer of bound proteins grows, the instrument detects that change in distance through optical interference between two reflection points: a reference layer near the sensor surface and the outer edge of the protein layer.

A spectrometer continuously monitors these reflections. After establishing a baseline measurement, the system tracks how the interference pattern shifts across the entire visible spectrum as the protein layer changes thickness. The instrument plots these changes on a graph called a sensorgram (a real-time binding curve), with time on the X-axis and “nm shift,” the measurable change in interference pattern, on the Y-axis.

Watch how BLI translates molecular binding into actionable data

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Gator Bio’s BLI Platform

Gator Bio’s BLI instruments allow users to analyze samples in standard 96-well or 384-well microplate formats, measuring up to 32 samples simultaneously. The plate-based design enables the instrument to move biosensors sequentially from well to well, capturing binding data at each step of your assay protocol. This high-throughput capability means you can screen hundreds of samples per day while maintaining the kinetic precision needed for confident decision-making.

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How BLI Measures Binding

BLI transforms binding responses into quantitative data through two primary approaches: measuring binding kinetics or measuring total binding response.

Quantitation Workflows

For concentration measurements, BLI monitors the initial rate of binding (nm shift per second), which is directly proportional to both the association rate constant (k_on) and analyte concentration. This relationship creates a concentration-dependent signal: higher concentrations produce steeper initial slopes.

To quantify unknown samples, you run a series of known standard concentrations to generate a calibration curve, then compare your unknowns against this standard curve. Because the analysis focuses on initial binding rates, these quantitation experiments are remarkably fast, often finished in less than 2 minutes per sample.

Rapid quantitation through initial rate analysis delivers results in under 2 minutes per sample

Kinetics Workflows
For kinetic characterization, BLI provides real-time measurement of both binding (association) and unbinding (dissociation) between biomolecule pairs. This allows you to determine three critical parameters through curve fitting: – k_on (association rate constant) – how fast molecules bind – k_off (dissociation rate constant) – how fast molecules unbind – K_D (equilibrium dissociation constant) – overall binding affinity.

For interactions that reach equilibrium quickly, you can use endpoint binding measurements to determine K_D . The minimal setup time and multi-channel format make BLI particularly attractive for high-throughput screening campaigns where you need to evaluate dozens or hundreds of candidates.

Real-time kinetics reveal the complete binding profile—from initial association through full dissociation

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Key Terms in BLI

Understanding BLI terminology helps you read and understand BLI literature:

• Capture Molecule: The reagent coated on the probe surface that is specifically designed to capture the first molecule of the binding pair
• Ligand: The first molecule of the binding pair captured onto the biosensor surface
• Analyte: The second molecule that binds to the immobilized ligand (your molecule of interest)
• Baseline: A buffer-only step that establishes the sensor’s stable starting point, allowing you to distinguish meaningful binding signal from instrument drift. A second baseline can also help to verify ligand binding to the biosensor.
• Loading: The kinetics assay step where biosensors are exposed to ligand-containing solution, allowing ligand to immobilize on the sensor surface
• Association: The kinetics assay step where analyte-containing solution contacts the ligand-loaded biosensor, enabling binding to occur
• Dissociation: The kinetics assay step where the biosensor returns to buffer-only conditions, allowing bound analyte to release from the sensor

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Assay Design Considerations

Successful BLI experiments require a thoughtful experimental design. Here are the most critical considerations:

Buffer Matching is Critical

BLI is an optical technique that responds to changes in the refractive index. Variations in buffer composition between assay steps can create signal artifacts that overwhelm your binding data. For optimal performance:

• Maintain consistent buffer conditions across all assay steps
• Watch for refractive index shifts from pH changes, DMSO, glycerol, sucrose, and other solvents
• Remember that glycerol isn’t volatile and may remain in lyophilized proteins
• For quantitation assays, pre-soak biosensors in buffer similar to your samples to minimize non-specific signal.

Choosing the Right Immobilization Strategy

Kinetics assays require immobilizing your ligand to the biosensor without blocking the analyte binding site. The most common strategies target conserved domains or protein tags:

Common capture approaches:

• Fc-binding biosensors: Protein A, Anti-Mouse Fc XT, Anti-Human Fc for antibodies
• Tag-based biosensors: Anti-His, Ni-NTA for His-tagged proteins; Strep-Tactin XT for twin-Strep-tagged proteins

• Biotin-Streptavidin: non-reversible, Streptavidin biosensors for biotinylated proteins (note: use a biotinylation protocol that limits the number of biotins per molecule – lysines are common in binding domains, and biotinylation can dramatically reduce analyte binding efficacy)

Fc-binding and Tag-based biosensors offer the advantage of regeneration: a simple low pH wash typically removes bound analyte, allowing biosensor reuse and reduced costs.

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Optimizing Analyte Concentration Range

Proper concentration selection is essential for accurate kinetic measurements. Your concentration series should:

• Upper range: Highest concentrations should show clear curve saturation (beginning to plateau)
• Lower range: Lowest concentrations should remain well below half-maximal binding
• Spacing: Use serial dilutions (typically 2-fold or 3-fold) to cover 2-3 orders of magnitude