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This page holds the standard methods for our BLI binding characterization assay. The concentration series, step timings and capture format for your experiment can differ, so check your Binding Data Package for the values used in your run. For the SPR equivalent, see SPR methods.

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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.

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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.

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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.

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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.

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Data processing and model fitting

Data were processed and fitted globally to a 1:1 Langmuir binding model using Adaptyv fitting software. All sensorgrams underwent standardized preprocessing and curve fitting. Preprocessing included trimming to the relevant phases (association, dissociation, baseline), correcting signal jumps at phase transitions, aligning association and dissociation phases, and subtracting baseline and reference signals. Fitting proceeded through multiple methods in a prioritized order. Initial fits were performed individually using global fitting, followed by full, dissociation-only or slope-based models. Where these were not possible, group-level models were applied: equilibrium (saturation), constant (flat) and semi-log linear (linear). Final kinetic parameters (kon, koff, KD) were selected based on fit quality. Global fitting was applied using a 1:1 model across all concentrations. In global mode, koff and KD were fitted directly, and kon was calculated as koff / KD. Fits were scored and filtered on quality metrics. Ligands were classified as binders or non-binders based on the presence of quantifiable binding curves and calculated KD 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.