Genepix personal 4100a

The viability of nanobiofilms on the microarray was determined by staining with FUN-1 fluorescent dye. Although originally developed for the visualization of metabolically active fungal cells (which are able to process the dye), FUN-1 also binds to the plasma membrane of viable and nonviable bacterial cells (24 (link)). The excitation and emission spectra of FUN-1, 480 to 535/550 nm, are compatible with the filters installed in most microarray scanners. Briefly, the nanobiofilms were stained with 1 μM FUN-1 by immersion of the entire microarray slide in a staining jar and incubation in the dark at 37°C for 30 min. Following incubation, the nBioChip microarray was washed in phosphate-buffered saline (PBS) by a simple dunk-and-rinse procedure to remove excess stain. After air-drying of the chip, a microarray scanner (GenePix Personal 4100A; Axon Instruments, Union City, CA) was used to scan for images. The microarray scanner images were analyzed with GenePix Pro V7 (Axon Instruments, Union City, CA) to determine the antimicrobial susceptibility profile, as described in references 12 (link) and 21 (link). Briefly, the percentage of viability of cells in response to different doses of different drugs was calculated from the fluorescence intensities of individual spots after setting the fluorescence intensities of live and dead controls at 100% and 0%, respectively.

Initially, 50 μl of sample was cultured on a fresh rabbit blood agar plate for 24 h. Growth was harvested for DNA extraction and analysis, as previously described [40 (link)]. In brief, microarrays were created by spotting oligonucleotide probes onto a glass slide using a SpotArray 7.2 (PerkinElmer). A two-step mPCR (to amplify the gene of interest and then label the PCR products) was conducted in two batches each. The resultant products were hybridised to a microarray slide for 16 h before being scanned (GenePix personal 4100A, Axon Instruments), and the signal intensity calculated with GenePix Pro 6.0.

Protoarray® v5.0 was obtained from Life Technologies (catalog number PAH0525101). Supplementary Table S1 contains an Excel file of Protoarray® v5.0 protein content. Protocols were followed according to the manufacturer's instructions (Protoarray® v5.0, Invitrogen, MA, USA). Slides were incubated in Protoarray® Synthetic Block for 1 h at 4°C with shaking at 50 rpm. During this time, reactions were prepared in a volume of 120 µl as follows: 25 or 50 nM of the purified SCF^Fbxo7 or Fbxo7(ΔF-box) in combination with ubiquitin mix [E1 (100 nM), UbcH5a (500 nM), Mg-ATP (2 mM), and biotin-ubiquitin 0.1 mg/ml in ubiquitination buffer; Boston Biochem]. The slides were washed with assay buffer (AB; 50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM MgSO₄, 0.1% Tween 20, 1% BSA, and 1 mM DTT) and 110 µl of the reaction was added to the slide and overlaid with a coverslip followed by incubation for 1.5 h at 30°C in a humidified chamber. Slides were washed in 0.5% SDS and AB and then incubated with 1 µg/µl of streptavidin–AlexaFluor 647 for 45 min at 4°C with shaking. The arrays were washed with AB, once with distilled water, and finally dried by centrifugation at 1000 × g for 2 min, before being scanned on a GenePix Personal 4100A (Axon–Molecular Devices).