Outer Membrane Vesicle Protein Quantification

A Bradford assay was used to determine outer membrane vesicle protein concentrations, as previously described (20 (link)). To generate a standard curve, bovine serum albumin (BSA) was diluted at 0 to 20 mg/mL in Pierce Coomassie Plus assay reagent (Thermo Fisher) to a final volume of 1 mL. Outer membrane vesicles were diluted at 2, 5, 10, 15, and 20 μL in reagent to a final volume of 1 mL. A microplate spectrophotometer (Fisherbrand AccuSkan) was used to measure the absorbance (OD₅₉₅) of the standard and samples in a 96-well plate (BrandTech). Protein concentrations were determined by comparing the optical densities of the samples to the standard curve plotted in Microsoft Excel, and final quantifications were graphed in GraphPad Prism 9. Experiments were reproduced three times from each outer membrane vesicle isolation, and one representative data set is reported.