Quantifying Curlin Protein Expression

Protein concentration from stool and brain samples was measured using the Bradford assay (Bio-Rad, Hercules, CA, USA). Thirty µg of protein was loaded and separated on a 10–12% SDS-PAGE gel and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Sigma). The membranes were blocked with 3% BSA in TBS Tween for 60 min at room temperature and incubated overnight at 4 °C with the primary antibody CsgA_ECO57 (major curlin subunit from E. coli) (Cusabio Technology LLC, Houston TX, USA.) at 1:3500. This antibody was previously validated [15 (link)]. After that, the membranes were incubated with the secondary antibody in the blocking buffer and blots were incubated with anti-rabbit (Abcam, AB6721) secondary antibody conjugated with horseradish peroxidase (1:20,000). GAPDH (1:40,000, ab181602) was used to normalize the data. Images were analyzed with ChemiDoc™ XRS + System Image Lab™ software (Bio-Rad, Hercules, CA, USA). The assays were performed three times using independent blots.