N ethyl n 3 dimethylaminopropyl carbodiimide

Surface plasmon resonance (SRP) experiments were performed on a Biacore X100 (GE Life Sciences, NJ) at 25.0 °C for determining Merlin binding to NHERF1. Before the binding experiments, the hydrogel matrix of the CM5 Biosensor chip (GE Life Sciences, NJ) was activated by N-hydroxysuccinimide and N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (GE Life Sciences, NJ). The ligand, which was 5 μg/ml NHERF1 was dissolved in 10 mM sodium acetate, pH 5.2, was injected to coat the activated surface. Uncross-linked ligand was washed away, and uncoated sites were blocked by 1 M ethanolamine, pH 8.5. The analyte, Merlin or Merlin(S518D), was dissolved in HBS-EP buffer containing 10 mM HEPES buffer, pH 7.4, 300 mM NaCl, 3 mM EDTA, and 0.005% surfactant polysorbate 20. The analyte was injected at a series of concentrations over the NHERF1-coated surfaces at 10 μl/min for 3 min. At the end of each injection, the sensor chip was regenerated with 4.0 M MgCl2, 50 mM triethylamine, pH 9.15, and HBS-EP buffer.