Fastprep 24 homogeniser

Escherichia coli cultures grown for 16 h were diluted into fresh 2YP media to an OD₆₀₀ of 0.03. For protein expression and conjugation, after 4 h of growth the media was supplemented with 0.5 mM IPTG and incubated at 28 °C for 24 h. Cultures were pelleted by centrifugation at 4000×g for 10 min 4 °C. At this stage pellets were either lysed for immunoblot or lipid extraction and silver stained as described previously [25 (link), 84 (link)].
Pellets from strains expressing polysaccharide only were resuspended in PBS to OD_600 nm of 10 with 1 mg ml-1 lysozyme and benzonase 40 U ml⁻¹ and boiled for 10 min before SDS-PAGE analysis. Conjugate samples were resuspended in 1.5 ml lysis buffer (50 mM NaH₂PO₄, 0.3 M NaCl and 10 mM imidazole, pH 7.5) with lysozyme and benzonase as above, and lysed using 5 × 40 s bead-beating rounds at 6.5 m/sec using a FastPrep-24 homogeniser (MP Biomedicals, Belgium), before His purification.

Streptococcus sanguinis whole-cell protein extracts were prepared using a FastPrep-24 homogeniser (MP Biomedicals) and quantified as described elsewhere (6 (link)). Separation of the proteins by SDS-PAGE, subsequent blotting to Amersham Hybond ECL membranes (GE Healthcare) and blocking were carried out using standard molecular biology techniques. To detect PilE1 and PilE2, we used previously described (6 (link)) primary rabbit antibodies (1/2000 dilution), an ECL HRP-linked anti-rabbit antibody (GE Healthcare) (1/10 000 dilution), and Amersham ECL Prime (GE Healthcare). To detect His-tagged proteins, we used a commercial HRP-linked anti-6His antibody (Sigma) at 1/10 000 dilution.

To analyse for ONNV RNA, the mosquito body parts were thawed at room temperature and homogenised in a FastPrep-24 homogeniser (Mpbiomedicals, USA) at 40 m/s for 20 s. The mosquito’s thorax and abdomen and the combined legs/wings/head samples were tested for ONNV RNA presence through RNA extraction using the QIAamp Viral RNA kit (Qiagen, Valencia, CA, USA) according to manufacturer’s protocol followed by q-RT-PCR. The mosquito saliva was tested for ONNV presence by cytopathogenic effect (CPE) on cell cultures followed by RNA extraction and qPCR. Vero B4 cells with a seeding density of 20,000 cells/well were seeded in a 96-well plate. When the wells had reached confluency, the cells were infected with 20 µl of saliva sample. The inoculated saliva samples were manually monitored for CPE daily for a week, and wells that showed CPE were harvested; RNA was extracted and q-RT-PCR performed. For the qPCR (Fisher Biosystems) we used Biosystems qPCRBIO Probe 1-step Go Lo-ROX kit and the following protocol: 1 cycle at 45 °C for 10 min, 1 cycle at 95 °C for 5 min and 40 cycles at 60 °C using primers targeting the ONNV Envelope genes E1 and E2 [24 (link)]. The specificity and sensitivity are shown elsewhere [24 (link)]. The probe was designed in close proximity to the forward and reverse primer and did not overlap with a primer binding site on the same site [24 (link)].