Centricon centrifugal filter with a mwco of 10 000

BLI experiments were performed using an Octet system (forteBIO) at room temperature (~23 °C). MBP was biotinylated using the biotination kit EZ-Link™ NHS-PEG4-Biotin (Thermo Fisher Scientific). Biotin label freshly dissolved in water was added to the protein solution to a final molar ratio of 1:1 in buffer containing 50 mM KPi, pH 7, 150 mM NaCl, 0.05% NaN₃, and the solution was mixed at room temperature for 45 min. Unlabeled biotin label was removed by extensive buffer exchange using Centricon® Centrifugal Filter with a MWCO of 10,000 (Millipore) at 4 °C using a buffer containing 20 mM HEPES (pH 7), 150 mM KoAc, and 0.05% NaN₃. Biotin-labeled MBP (200 nM) denatured in 8 M urea was immobilized onto the Streptavidin (SA) biosensor, and the biosensors were subsequently blocked with Biocytin in 8 M urea solution before a quick 30 s dip into the urea-free buffer. SecB or TF previously diluted was applied in a dose dependent manner to the biosensors immobilized with MBP. Bovine serum albumin (BSA) powder (Sigma-Aldrich) was added to a final concentration of 2% to avoid nonspecific interaction. Parallel experiments were performed for reference sensors with no MBP captured and the signals were subsequently subtracted during data analysis. The association and dissociation periods were set to 2 min and 5 min, respectively.