Goat α rabbit 680 nm

Whole cell bacterial lysates were prepared for Western blot analysis as follows. Bacteria were harvested from overnight growth on CFA agar with or without 200 µM DFOM in PBS, and the OD₆₀₀ was used to normalize to the samples for bacterial numbers (55 (link)). The normalized suspensions were then mixed 1:1 with 2× Laemmli Sample Buffer (Bio-Rad). Proteins were separated on a 12% Mini-Protean TGX Precast Gel (Bio-Rad) and transferred to PVDF membrane. The membrane was blocked in a 10% (wt/vol) nonfat milk buffer in PBS and incubated with absorbed polyclonal Rabbit α-CS14 (Rockland Immunochemicals). The secondary antibody was Goat α-Rabbit 680 nm (Invitrogen), and proteins were visualized using the LI-COR Odyssey Laser Scanner. Additional Western blots of CFA agar with or without 200 µM DFOM grown strains were performed and stained with Rabbit anti-DnaK (Invitrogen) and Goat α-Rabbit 680 nm (Invitrogen) antibodies to confirm equal loading.

Whole cell bacterial lysates were prepared for western blot analysis as follows. Bacteria were harvested from overnight growth on CFA agar plates or in Terrific broth static culture into PBS, and the OD₆₀₀ was used to normalize the samples for bacterial numbers. The normalized suspensions were then mixed 1:1 with 2x Laemmli Sample Buffer (Bio-Rad). Proteins were separated on a 12% Mini-Protean TGX Precast Gel (Bio-Rad) and transferred to PVDF membrane. The membrane was blocked in a 10% (w/v) nonfat milk buffer in PBS and incubated with absorbed polyclonal Rabbit α-CFA/I (Rockland Immunochemicals) or polyclonal Rabbit α-LngA antibodies (Genscript). The secondary antibody was Goat α-Rabbit 680 nm (Invitrogen), and proteins were visualized using the LI-COR Odyssey Laser Scanner. An identical 12% Mini-Protean TGX Precast Gel (Bio-Rad) was run with each Western blot and stained with GelCode Blue Stain Reagent (Thermo Scientific) to determine the total protein in each lane. Additional Western blots of CFA agar grown and Terrific broth grown strains were performed and stained with anti-DnaK (Invitrogen) and Goat α-Rabbit 680 nm (Invitrogen) antibodies to confirm equal loading. Fiji (ImageJ) was used to quantify the amount of protein (ng) expressed by each sample within individual Western blots as compared to the protein concentration of the purified fimbrial control.

Whole cell bacterial lysates were prepared for western blot analysis as follows. Bacteria were harvested from overnight growth on agar plates into 1xDPBS (Corning, Corning, NY) and the OD₆₀₀ was used to normalize to an OD of 3.0. The normalized suspensions were then mixed 1:1 with 2x Laemmli Sample Buffer (0.125 M Tris pH 6.8, 4% SDS, 20% Glycerol, 0.2% Bromophenol Blue, 10% β-mercaptoethanol). Proteins were separated on a 12% Mini-Protean TGX Precast Gel (Bio-Rad, Hercules, CA) before being transferred to a PVDF membrane. The membrane was blocked in a 10%(w/v) nonfat milk buffer in 1xDPBS and then incubated with polyclonal Rabbit α-CS6 sera (Rockland Immunochemicals, Limerick, PA) in 10% (w/v) nonfat milk buffer overnight at 4°C on an orbital shaker. Following washing, the membrane was incubated with secondary antibody Goat α-Rabbit 680 nm (Invitrogen, Carlsbad, CA) and proteins visualized using the 700 nm wavelength setting on an LI-COR Odyssey Laser Scanner.