Mouse anti c myc

In case of surface displayed PCNA surface accessibility was shown by proteinase K digestion and outer membrane and periplasmatic protein isolation as described by Park et al. (2015 (link)). The outer membrane protein isolation samples were separated by SDS‐PAGE and analysed by Western blot analysis as described by Gercke et al. (2021 ). A primary Anti‐myc antibody (mouse anti‐c‐myc, 1:1500 in PBS containing 1% BSA, Thermo Fisher Scientific, Braunschweig, Germany) was used instead of the anti‐His₆ antibody described by Gercke et al.

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.