Octet red384

ForteBio Octet Red384 was used to determine the binding kinetics initially using seven concentrations of the DARPins from 3,000 nM to 4.1 nM in 1/3 dilution steps in buffer (PBS containing 1 mg ml⁻¹ BSA and 0.01% Tween), with varying concentrations for repeat determinations. For each sensor the following protocol was carried out: Baseline wash in octet buffer for 60 s, loading of biotinylated Ras (1–166; mutant or wild type; GDP or GTPγS) at 5 μg ml⁻¹ for 180 s followed by a further 60 s baseline wash. DARPins were then associated for 180 s and dissociated for 600 s by washing in buffer. For each sample a reference well was included only containing buffer. Data were analysed using ForteBio data analysis. Reference wells were subtracted from sample wells and 1:1 global fitting model was used to fit curves to sensorgrams and determine K_on, K_off and K_d values. For measurements of kinetic constants for Ras GDP forms, 2 mM MgSO₄ and 100 μM GDP were included in the buffers. For measurements of kinetic constants for Ras GTPγS forms, 2 mM MgSO₄ and 100 μM GTPγS were included in the buffers.

The OctetRED₃₈₄ (ForteBio Inc) was used for label-free measurements of binding as detected on biosensors as a function of optical thickness (nm) versus time as described in⁵⁵ . All assay steps were performed at 25 °C with 1000 RPM stirring in tilted-bottom 384-well plates (ForteBio Inc) containing 80 μL sample volume. PBS (pH 7.4) containing 0.1% Tween 20 and 10 mg/mL bovine serum albumin (BSA) was used as the kinetic assay buffer. For kinetic determination, approximately 0.5 nm optical thickness Fab was loaded onto Protein L biosensors. Next, an association phase was performed over a range of RAD51 concentrations for 2–3 minutes. Biosensors were then moved to kinetics buffer alone to measure the rate of dissociation. Association and dissociation rates and dissociation constants were calculated using ForteBio Data Analysis version 7.1 curve-fitting software with a 1:1 Langmuir binding model. A reference well with buffer alone was subtracted from all values to account for sensor drift.
To calculate the IC₅₀ for Fab inhibition of RAD51 DNA binding, 1 nm optical thickness of 5′-biotinylated oligo(dT)₃₆ was loaded onto a streptavidin biosensor from a 1 μM solution. Association to 0.5 μM RAD51 in the absence of Fab-F2 was performed to yield maximal binding at equilibrium, from which the relative binding at equilibrium of parallel wells containing Fab-F2 could be subtracted.

BLI data were measured using an Octet RED384 (ForteBio) instrument using ForteBio Octet data acquisition software (v12.0.2.11). Biotinylated antigens were immobilized on a streptavidin biosensor and loaded until 0.4-nm signal was achieved. After blocking with 10 µM biotin, purified antibodies in solution were used as the analyte. PBS + 0.05% Tween + 0.2% bovine serum albumin (BSA) was used for all buffers. Data were analyzed using the ForteBio Octet data analysis software (v12.0), and kinetic parameters were determined using a 1:1 monovalent binding model.