Typhoon trio imager

Urea polyacrylamide gel (urea PAGE) and agarose electrophoresis (AE) assays were performed to test the endonucleolytic reaction in the micromolar regime of substrate. 20-nt RNA or 19-nt, 30-nt and 66-nt DNA were incubated with PA-Nter in the presence of 0.25–1 mM Mg²⁺ or Mn²⁺ at 25°C for 1 h. Concentrations of reagents are specified in figure legends. Next, samples were denatured by adding 1 μl of 40 mM EDTA in formamide and incubation at 70°C for 5 min. Control time course cleavage was done by stopping the reaction in 10 min steps. Urea polyacrylamide gel (20%) was pre-run in TBE buffer (200V) for 30 min. Subsequently, samples were loaded and run for 2 h. M13mp18 plasmid was incubated with PA-Nter in the presence of 0.25–1 mM Mg²⁺ or Mn²⁺ at 37°C for 2 h and inactivated in 80°C for 20 min. Samples were loaded onto 1% agarose gel and run in TAE buffer (90 V) for 45 min. Gels were stained with ethidium bromide and distained with distilled water for 15 min, respectively, next trans-illuminated by ChemiDoc™ MP System (Bio-Rad) and analysed in ImageJ (http://imagej.nih.gov/ij/). Fluorescent images of gels showing the cleavage of 30-nt fluorescent DNA hairpin were registered with Typhoon Trio imager with default settings (GE Healthcare).

One hundred μg of proteins were rehydrated on 11 cm, pH 4–7 linear IPG strips and then subjected to isoelectric focusing (IEF) using a Protean IEF Cell (Bio-Rad, Hercules, CA) using the conditions 250 V for 20 min, 8,000 V for 2 h and then 8,000 V till total volt hours reached 40,000. After IEF, the strips were reduced and alkylated sequentially for 15 min. The second-dimension SDS-PAGE was performed using 4–20% in a Criterion polyacrylamide gradient gels (Bio-Rad, Hercules, CA) for 2 h at 100 V. The 2-D gels were fixed in 50% methanol and 10% acetic acid and then stained with fluorescent SYPRO Ruby protein gel stain and scanned using Typhoon Trio Imager (GE Healthcare, Piscataway, NJ, USA). The 2D resolved total proteome gels were compared using the PD Quest^TM Advanced 2-D Analysis software, Version 8.0.1 (BioRad, Hercules, CA). The 2D gels were also used for Western blot analysis. The proteins resolved on 2D gels were transferred to a nitrocellulose membrane and the membrane was incubated with Ehrlichia specific polyclonal sera or with p28 OMP monoclonal antibody, 18.1 (Winslow et al., 2000 (link)). Polyclonal sera were obtained from wildtype C57BL6 mice infected with E. chaffeensis to detect immunological proteins (Ganta et al., 2004 (link)). The membranes were developed using X-ray developer.

Translation mixtures (TM) of synthesized Mel-Glyco-eYFP-His, ET-B-GFP and ET-B-eYFP were centrifuged at 16.000 × g for 10 min at 4 °C. The pellets were resuspended in PBS (phosphate-buffered saline, PBS Dulbecco without Ca²⁺ and Mg²⁺, Biochrom AG) and treated for 10 min with 0–0.1% polyoxyethylen(23)laurylether (Brij 35; Sigma Aldrich). To analyze the perforation of the microsomal membranes, the resuspended and detergent-treated pellet was centrifuged again at 16.000  ×  g for 10 min at 4 °C and divided into supernatant (S) and vesicular fraction (VF). Translation mixture, supernatant and vesicular fraction were applied to microwell glass slides (µ-Slide, 18 well, Ibidi) and cell-free synthesized eYFP-labeled proteins were excited at 488 nm. Fluorescence signals were recorded at 526 nm using a phosphorimager system (Typhoon TRIO + Imager, GE Healthcare).

RNA degradation assays were performed in buffer that contained 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 1 mM MgCl₂, and 1 mM ATP at 30 °C with 60 nM protein and 30 nM RNA. The reaction was stopped at the selected time-points by the addition of loading buffer (95% formamide and 20 mM EDTA). Reaction products were resolved by 20% denaturing PAGE and visualized with Typhoon Trio imager (GE Healthcare). Cg-Dss1 or Cg-mtEXO activity was quantified in ImageQuant TL 7.0 by dividing the amount of the final cleavage product by the total RNA signal from each lane. In the anti-fluorescein antibody protection experiment, the Monoclonal Anti-Fluorescein (FITC) IgG CF™ 488 A antibody (Sigma-Aldrich) was incubated with the 5′-fluorescein-labeled RNA substrate for 5 min at a final concentration of 2 μg ml⁻¹ prior to the degradation assay, which was conducted as described above. A list of substrates that were used for the activity tests and their sequences are presented in Supplementary Table 2.