Bio dot dot blot apparatus

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.

Dot-blot analysis for screening disease resistance was performed following Sahu et al. [12 (link)]. Total genomic DNA was denatured by adding an equal volume of 0.6 M sodium hydroxide and an equal amount of each denatured DNA (~ 1 μg) was spotted onto three Hybond N+ membranes (Amersham Bioscience) embedded in a BIO-DOT dot-blot apparatus (Bio-Rad). The samples were spotted in 96-well formats to prepare three identical arrays. The membranes were hybridized with α³²P-dCTP labeled DNA A specific AC1 gene sequence, and then neutralized with neutralization buffer (0.5 M Tris-Cl, pH 7.4, 1.5 M NaCl) for 3 min, washed with 2% standard saline citrate (SSC) and cross-linked using UV cross-linker (Stratagene). Radiolabeled probe was made by random primer labeling method by using Megaprime DNA labeling system (Amersham Biosciences) [12 (link)].