Chemidoc itts2 imager

The PVDF membrane with transferred proteins was briefly stained with Ponceau S to observe successful protein transfer and destained with water before proceeding to antibody incubations. The membrane was blocked overnight in OneBlock Western CL blocking solution (Genesee Scientific; San Diego, CA, USA) at 4 °C prior to incubation in primary antibody for 1–2 h at room temperature on a rocker. Two brief rinses and three 5-min washes with Tris-buffered saline with Tween 20 (TBST) were performed, and the membrane was then incubated in secondary antibody for 1 h at room temperature on a rocker. The washes were repeated, with the final wash replaced by Tris-buffered saline (without Tween, TBS). The membrane was incubated in Luminata Forte HRP Chemiluminescence developer (Millipore Sigma) for two minutes prior to visualization on the ChemiDoc-ItTS2 Imager (UVP; Upland, CA, USA). All antibodies were diluted in OneBlock Western CL (Genesee Scientific; San Diego, CA, USA). Primary antibodies were from rabbit and developed to react to human sera, and secondary antibody was goat anti-rabbit conjugated to horseradish peroxidase (Table 2). Secondary-only controls were run for each of the three protein targets to ensure specificity; no bands appeared on these controls. Each sample for each protein was blotted in triplicate.

To detect EBOVpp production, purified EBOVpp were processed using RIPA buffer (Sigma) supplemented with cOmplete Mini Protease Inhibitor Cocktail (ROCHE; Basel, Switzerland) and the protein concentrations were determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories; Hercules, CA, USA). Following which, samples were resolved by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto a nitrocellulose membrane by standard Western blotting techniques. The blots were then probed using the primary anti-EBOV GP (subtype Zaire, strain Mayinga 1976) rabbit polyclonal antibody (Sino Biological; Beijing, China) at 1:1000 dilution, and HIV-p17 monoclonal antibody (Santa Cruz Biotechnology; Dallas, TX, USA) at 1:200 dilution, followed by the respective horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam; Cambridge, United Kingdom) at 1:3000 dilution and detection using ECL chemiluminescent substrate (Bio-Rad Laboratories). Images were taken with a ChemiDoc-It^TS2 imager (UVP; Upland, CA, USA).

PCR with species-specific primers was used to identify the four bacterial species from isolates obtained from beef cattle, and to confirm the biochemical (aerobic) and morphologic (Mycoplasma) identification of the isolates from the dairy cattle. Primers were designed in Primer3 (Koressaar and Remm 2007 (link); Untergrasser et al. 2012 (link)) to targeted genes specific to each species (Table 3). PCR protocols for M. haemolytica (Alexander et al. 2008 (link)) and P. multocida (Ullah et al. 2009 ) were performed as described previously. Protocols for H. somni and M. bovis were developed during this study. PCR was performed on an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) with 1× GoTAQ Green Master Mix (Promega Biosciences LLC, San Luis Obispo, CA), 10 μM of each primer and 10 ng of DNA for 5 min at 95°, 35 cycles of 30 sec at 95°, 30 sec at 56°, and 45 sec at 72°, followed by 10 min at 72°. Products were visualized on a 1.5% agarose gel using a ChemiDoc-It^TS2 Imager (UVP, LLC, Upland, CA), purified using the QIAquick PCR Purification Kit (Qiagen), Sanger sequenced (www.davissequencing.com) and aligned to their respective reference genomes using Bowtie2–default v2.2.8 (Langmead and Salzberg 2012 (link)) to verify that the appropriate target regions were amplified.