> ## Documentation Index
> Fetch the complete documentation index at: https://docs.adaptyvbio.com/llms.txt
> Use this file to discover all available pages before exploring further.

# BLI methods

> Standard methods for Adaptyv BLI binding characterization on Gator Bio instruments

This page holds the standard methods for our [BLI](/wiki/technologies/BLI) binding characterization assay. The concentration series, step timings and capture format for your experiment can differ, so check your [Binding Data Package](/docs/experiment-setup/binding-data-package) for the values used in your run.

For the SPR equivalent, see [SPR methods](/wiki/methods/spr-methods).

## Cell-free expression of protein ligands

DNA constructs encoding the ligands, each fused to a C-terminal assay tag, were designed by reverse-translating the target protein sequences. The sequences were optimized for manufacturability and yield using codon optimization algorithms to maximize expression efficiency. The optimized DNA constructs of the variants and the 3′ fragment containing the linker and affinity tags were ordered as Gene Fragments from Twist Bioscience.

Constructs were assembled using the NEBuilder HiFi DNA Assembly Kit (NEB) in 2 μL reactions. The assembled products were characterized by capillary electrophoresis (Agilent ZAG DNA Analyzer, ZAG-135-5000 Kit, FA/ZAG 96-Capillary Array Short, 33 cm), and their concentrations were measured using the Qubit DNA Quantification Kit (Invitrogen). Ligand expression was carried out in 8 μL reactions using an optimized prokaryotic in vitro translation system and 4 nM of assembled gene fragment. Reactions were incubated at 37 °C for 8 hours. Post-expression, protein concentration and yield were normalized using an affinity-based quantification assay.

## Affinity characterization by biolayer interferometry (BLI)

Kinetic binding measurements were performed on a BLI instrument (Gator Bio) using Strep-Tactin XT probes to capture Twin-Strep-tagged ligands.

### Sensor preparation and capture

Probes were equilibrated in running buffer before use. Ligands were captured with the following sequence:

* **Baseline 1:** 120 s in running buffer
* **Ligand loading:** 120 s (target loading shift 0.5–1.0 nm)
* **Baseline 2:** 200 s in running buffer

Running buffer was 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.2% Tween-20, pH 7.4. Temperature was held at 25 °C and data were sampled at 5 Hz.

### Association and dissociation (multi-cycle kinetics)

Antigen solutions were prepared in running buffer as a half-log dilution series of 4 concentrations (1000 nM to 30 nM). Each cycle consisted of:

* **Association:** 220 s in antigen solution
* **Dissociation:** 240 s in running buffer

Between cycles, probes were regenerated in 10 mM glycine-HCl, pH 1.5, applied 5 times for 10 s each, followed by neutralization in running buffer. Negative controls (buffer-only and a non-binding ligand) were included for reference subtraction and drift correction.

### Data processing and model fitting

Data were processed with Adaptyv fitting software. All sensorgrams underwent standardized preprocessing: trimming to the relevant phases (baseline, association, dissociation), correcting signal jumps at phase transitions, aligning the association and dissociation phases, and subtracting baseline and reference signals.

Each design was fitted as a group: all of its association and dissociation curves across the concentration series were fitted simultaneously, with k<sub>on</sub> and k<sub>off</sub> shared across concentrations and R<sub>max</sub> fitted per curve. The default model is a 1:1 Langmuir interaction, and K<sub>D</sub> was calculated as k<sub>off</sub> / k<sub>on</sub>, with 95% confidence intervals for each parameter. Alternative binding models (bivalent analyte and conformational change) were also fitted. Where one described the curves better than 1:1, it was selected and its apparent K<sub>D</sub> reported; the portal shows this value, and the API returns it as `kd_app` with the model name in `binding_model`.

Candidate fits were filtered on quality before selection. A fit was kept only if its residual error in both the association and dissociation phases, normalized to signal size, stayed below a fixed threshold, its R<sub>max</sub> was consistent across concentrations, and bulk refractive-index jumps were small relative to the binding signal. The remaining fits were ranked by residual error, then penalized when a rate constant sat at the edge of what the assay can resolve, then ranked by confidence-interval width.

For how to read the result, with examples of good and poor fits, see [Judging fit quality](/guides/binding-characterization#judging-fit-quality) and [Common curve shapes](/guides/binding-characterization#common-curve-shapes) in the binding characterization guide.

Ligands were classified as binders or non-binders based on the presence of quantifiable binding curves and calculated K<sub>D</sub> values. Where a ligand produced a significant signal shift during the association phase (≥300% over the negative control) but the signal could not be reliably fit, the binding label was assigned based on the magnitude of the observed shift.

## Related pages

* [BLI](/wiki/technologies/BLI)
* [SPR methods](/wiki/methods/spr-methods)
* [Binding](/docs/experiment-types/binding)
* [Binding Data Package](/docs/experiment-setup/binding-data-package)


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