Total protein extracts from purified OMVs were prepared as described above. 20 μL aliquots were mixed with Laemmli SDS sample buffer (Alfa Aesar, Thermo Fisher Scientific, Waltham, MA, USA), boiled at 100 °C for 5 min and run on 10% SDS-PAGE in parallel with molecular weight markers (Page Ruler; Thermo Fisher, Waltham, MA, USA). Gels were stained with Coomassie Blue (0.1%). OMV production was quantified on the basis of OmpF/C band using the densitometry tool of the Image Lab software (Bio-Rad, Hercules, CA, USA), normalizing against the wt strain.
For immunoblot analysis, SDS-PAGE profiles were transferred to PVDF membranes (Hybond-P, Millipore, Tullagreen, Carrigtwohill, Ireland). The membranes were incubated overnight at 4 °C in a 1:50,000 dilution of a E. coli-OmpA rabbit antibody according to Ambrosi et al. [37 (link)], while the anti-rabbit secondary antibody was used in a 1:10,000 dilution. The protein-antibody complex was detected by enhanced chemiluminescence (Euroclone, Pero, Mi, Italy), as previously described [38 (link)]. The same densitometry procedure was followed to estimate OmpA abundance in the mutant strains, normalizing against the wt strain.
For immunoblot analysis, SDS-PAGE profiles were transferred to PVDF membranes (Hybond-P, Millipore, Tullagreen, Carrigtwohill, Ireland). The membranes were incubated overnight at 4 °C in a 1:50,000 dilution of a E. coli-OmpA rabbit antibody according to Ambrosi et al. [37 (link)], while the anti-rabbit secondary antibody was used in a 1:10,000 dilution. The protein-antibody complex was detected by enhanced chemiluminescence (Euroclone, Pero, Mi, Italy), as previously described [38 (link)]. The same densitometry procedure was followed to estimate OmpA abundance in the mutant strains, normalizing against the wt strain.
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