Anti mouse dylight 633

Detection of passenger surface display was performed using antibodies against the incorporated c‐myc epitope by flow cytometry. E. coli BL21 (DE3) cells containing plasmids encoding for autotransporter fusion proteins were cultivated up to an OD_578nm of 0.5. After the induction of gene expression, a part of the cells was incubated with proteinase K at 37°C for 1 h. The digestion of surface displayed peptides was stopped by the addition of 5 mM PMSF. Afterwards, the cells were washed three times with cold PBS and blocked with 5% bovine serum albumin (BSA) in PBS at RT for 15 min. Subsequently, 100 μL of the cell suspension (OD_578nm of 5) were incubated with the primary antibody (mouse anti‐c‐myc, 1:100 in PBS containing 1% BSA, Thermo Fisher Scientific, Braunschweig, Germany) at 37°C for 1 h. After further washing steps, the cells were incubated with the secondary DyLight633 conjugated antibody (goat anti‐mouse DyLight633, 1:50 in PBS containing 1% BSA, Thermo Fisher Scientific, Braunschweig, Germany) at 37°C for 1 h. Cells were washed three times with particle‐free PBS (0.22 μm) and stored on ice until they were analysed. Fifty thousand cells from each sample were measured by flow cytometry (FACS Aria III, BD Biosciences, Franklin Lakes, USA) using an excitation wavelength of 633 nm and BP 660/20 nm detection filters.