Nsb reducer

Kinetic titration series were performed in the interaction buffer (PBS with 0.5% Polysorbat 20, 0.1% BSA and 10% NSB reducer from GE Healthcare). 2.4 µM scFv IC16 was diluted four times with a dilution factor of two. To measure the interaction between Aß(1–42) and scFv IC16, the association and dissociation times were 270 and 90 s, respectively, for every analyte concentration. Further steps are comparable with “Parallel sensor kinetics of GB1 with biolayer interferometry” (Data Analysis software: 8.0.0.35). The sensorgrams with the concentrations: 0.24, 0.12, 0.06, 0.03 and 0.015 µM were fitted with the BiaEvaluation software 4.1 from Biacore using a 1∶1 binding model without RI-term.

USP4 CD, insert and D1D2 were biotinylated on lysines using half molar amounts of EZ-Link Sulfo-NHS-LC-LC-biotin (Thermo Scientific). SPR was performed at 25 °C on a Biacore T100 (GE Healthcare) using a SA sensor chip loaded with ~100 response units of biotinylated USP4. Concentration series of USP4 DUSP–Ubl in running buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 5 mM DTT and 0.05% Tween 20) supplemented with NSB reducer (GE Healthcare) were flowed over the chip at 30 μl min⁻¹. The program EvilFit was used to calculate populations of dissociation constants (K_d) with similar values for dissociation rates (k_off) allowing to remove the contribution of nonspecific accumulation (slow k_off component) to the overall dissociation constant.