Biacore t200 evaluation software 2

Experiments were performed at 25 °C with a Biacore^™ T200 apparatus (Biacore^™, GE Healthcare Life Sciences, Uppsala, Sweden). The experiments were performed on CM5 sensor chips (Biacore^™) coated with 140 resonance units (RU) of FasL (Peprotech). A flow cell left blank was used for double-referencing of the sensorgrams. Soluble full-length extracellular domain of CD95 (sCD95) or Δ58 truncated constructs (sCD95-Δ58) were prepared in the running buffer, 10 mM Na₂HPO₄, 150 mM NaCl, 3 mM EDTA and 0.05% Tween-20 at pH 7.4 and injected in triplicate at 25 µl/min. The regeneration of the functionalized surface was achieved with a 1-min pulse of 0.5% SDS. The sensorgrams were processed using Biacore T200 Evaluation Software 2.0 (Biacore^™). The association and dissociation rate constants, k_a and k_d, were determined by direct curve fitting of the sensorgrams to a Langmuir 1:1 model of interaction. The dissociation equilibrium constant, K_D, was calculated as k_d/k_a.