Biacore t100 instrument

The binding interaction between different compounds and Robo1 was examined by SPR using a Biacore T100 instrument (Biacore Inc., GE Healthcare, USA). Robo1 was immobilized on a CM5 sensor chip (Biacore Inc., GE Healthcare) by standard amine coupling using an amine coupling kit. The surface was activated using freshly mixed N-hydroxysuccimide (NHS; 100 mM) and 1-(3-(dimethylamino)propyl)-ethylcarbodiimide (EDC; 391 mM) (1/1, v/v) in water. Next, Robo1 (100 μg/mL) in aqueous NaOAc (10 mM, pH 5.0) was passed over the chip surface until a ligand density of approximately 2800 RU was achieved. The remaining active esters were quenched by aqueous ethanolamine (1.0 M, pH 8.5). The control flow cell was activated with NHS and EDC followed by immediate quenching with ethanolamine. HBS-EP (0.01 M HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% polysorbate 20; pH 7.4) was used as the running buffer for the immobilization and kinetic studies. A serial dilution of each compound in HBS-EP buffer and a 30 μL/min flow rate were employed for association and dissociation at a constant temperature of 25 °C. The surface was regenerated by a 30 s injection of aqueous NaCl (2.0M) at a flow rate of 30 μL/min. Data were fitted to a 1:1 binding model using BIAcore T100 evaluation software to obtain the equilibrium constant (K_D) data.

Measurements were done in USC NanoBiophysics Core Facility on a Biacore T100 instrument (Biacore). H-Ras(G12V) was immobilized on streptavidin sensor chips at the indicated surface density. Ras protein was exchanged with GppNHp on a chip where indicated by injecting SPR exchange buffer [50 mM Tris–HCl (pH 7.5), 1 mM EDTA, and 0.5 mM GppNHp] at 35 μL/min for 10 min. A concentration series of maltose binding protein–intrabody fusion was injected at a flow rate of 100 μL/min at 25 °C in SPR run buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.01% (vol/vol) Tween-20, 5 mM MgCl₂, and 10 μM GppNHp]. Data were analyzed by Biacore T100 Evaluation Software.