Octet red96 system

Biolayer interferometry (BLI) assays were carried out using an Octet RED96 system (ForteBio) at 28° C. BLI assays utilized a five-step protocol: equilibration (180 s), peptide ligand loading (20 to 60 s), baseline establishment (60 s), analyte association (various times), and analyte dissociation (various times). The analyte proteins (PCNA, Rad6-Rad18, or Rad6) were in BLI buffer (40 mM HEPES, pH 8, 150 mM NaCl, 0.5% bovine serum albumin, 0.002% Tween, and 5 mM 2-mercaptoethanol). The equilibration, baseline-establishment, and analyte-dissociation steps all used BLI buffer without analyte proteins. Prior to the experiment, the Streptavidin (SA) Biosensors (ForteBio) were soaked in BLI buffer for 10 min. Experiments were carried out with seven biosensors incubated with BLI buffer containing different concentrations of analyte. One biosensor was used as a reference and was incubated with BLI buffer without analyte. Control experiments were carried out with sensors that were not loaded with peptide ligands to assess whether there was any non-specific analyte binding to the biosensors. The response units from non-specific binding was subtracted from the response units for the experiments that displayed non-specific binding. Data was processed with the ForteBio Data Analysis software and Prism (GraphPad).

Protein–protein interactions were measured by using an Octet RED96 System (ForteBio) using streptavidin-coated biosensors (ForteBio). Each well contained 200 μL of solution, and the assay buffer was HBS-EP+ buffer (GE Healthcare Life Sciences, 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20) plus 0.5% non-fat dry milk blotting grade blocker (BioRad). The biosensor tips were loaded with analyte peptide or protein at 20 μg mL⁻¹ for 300 s (threshold of 0.8 nm response), incubated in HBS-EP+ buffer for 60 s to acquire the baseline measurement, dipped into the solution containing cage and/or key for 1800 s (association step) and dipped into the HBS-EP+ buffer for 1800 s (dissociation steps). The binding data were analysed with the ForteBio Data Analysis Software version 9.0.0.10.

BioLayer Interferometry (BLI) measurements of affinity K_D were obtained using the ForteBio Octet RED96 system as previously described [25 (link)]. Briefly, Protein A-coated biosensor tips were “activated” by incubation with 41nM TZ47-Fc, NKp30-Fc, or CC3-Fc for 10 min to load tips. Binding to soluble B7H6-His were assessed by allowing TZ47-Fc, NKp30-Fc, or CC3-Fc coated tips to equilibrate in PBS + 0.1% Tween-20 (PBST) for 5 min, followed by association steps in 200 μL of each antigen at concentrations ranging from 0 to 3000 nM for 10 min, and dissociation steps in 200 μL of PBST for 5 min. Data was analyzed using Octet^® System Data Analysis software.