Licor odyssey fc imaging system

Total bacterial lysates were electrophoresed on 12.5% SDS-PAGE gels and transferred onto nitrocellulose membrane (Invitrogen) for immunoblotting. Rabbit anti-PA4635 were used at 1:2,000 dilution and Mouse anti-EF-Tu antibody (Ball et al., 2016 (link)) was used at 1:20,000 dilution. Anti-rabbit or anti-mouse antibodies (dilution 1:30,000) labeled with fluorescent dye DyLight 800 (Thermoscientific) were used for the detection of PA4635 and EF-Tu using LICOR Odyssey Fc Imaging System (excitation 783 nm and emission 797 nm) to quantify the amount of proteins.

Cocultures were performed in triplicate in 150 μl of DMEM with 1 g/liter glucose in a polypropylene 96-well plate (Nunc). WT or mutant EHEC bacteria were inoculated 1:100 from overnight cultures grown in LB medium, and WT or mutant E. faecalis bacteria were inoculated 1:100 from overnight cultures grown in BHI medium. Plates were grown for 6 h in a 37°C, 5% CO₂, cell culture incubator in a water bath. Plates were then heated to 95°C for 10 min to kill bacteria. A standard curve of purified recombinant EspB protein was included so that absolute EspB concentrations could be determined. Heat-inactivated samples (100 μl) and standards were applied to a 0.2-μm-pore-size nitrocellulose membrane using a Bio-Dot dot blot apparatus (BioRad) according to manufacturer’s instructions. EspB protein was detected using a rabbit anti-EspB polyclonal antibody (1:10,000) and a goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (1:20,000). Dot blots were developed using SuperSignal West Pico Plus chemiluminescent substrate (Thermo) and imaged on a LiCor Odyssey FC imaging system. Intensity of the spots was quantified using ImageJ.

Samples were electrophoresed on 4–12% pre-cast ExpressPlus^TM PAGE Gels (Genscript) and transferred onto nitrocellulose membrane (Invitrogen) for immunoblotting. Rabbit anti-MgtC^{6 (link)} and rabbit anti-MgtB antibodies were used at 1:500 and 1:1000 dilution respectively. Mouse anti-DnaK antibody (Tebubio) was used at 1:5000 dilution, whereas mouse anti-T18 antibody (monoclonal antibody sc-13582, Santa-Cruz) was used at 1:1000 dilution. Rabbit anti-OmpF and anti-OmpC antibodies were used at 1:1000. Anti-rabbit antibody conjugated to horseradish peroxidase (Sigma) was used at 1:5000 dilution for the detection of MgtC, using Western Lightning® Plus-ECL (PerkinElmer Millipore). Anti-mouse or anti-rabbit antibody labeled with fluorescent dye DyLight 800 (Thermoscientific) were used for the detection of T18, DnaK, MgtB, OmpF and OmpC using LICOR Odyssey Fc Imaging System (excitation 783 nm and emission 797 nm) to quantify the amount of protein levels.