Vacuum centrifuge

EPO was prepared in a batch mode in a final volume of 100 µL. After each translation cycle, the original set of microsomes was collected by centrifugation (20 min, 15,000 × g). The microsomal pellet resulting from centrifugation was resuspended in a new aliquot of translation mixture containing fresh components required for translation, such as a fresh mRNA aliquot and fresh lysate deprived of microsomes by a centrifugation step (10 min, 4 °C, 16,000 × g). Each cycle corresponded to the incubation period of 90–120 min at 27 °C and 500 rpm in a thermomixer (Thermomixer comfort, Eppendorf, Hamburg, Germany). After the third cycle, vesicles were collected by centrifugation (20 min, 15,000 × g) and the pellet was subjected to a standard acetone precipitation protocol for 1 h to remove salts. Precipitated proteins were recovered by centrifugation (20 min, 15,000 × g) and were briefly dried in a vacuum centrifuge (Eppendorf, Hamburg, Germany).

Dried protein samples were processed as described previously.⁵⁴ (link) Briefly, protein pellets were dissolved in 50 mM ammonium bicarbonate buffer (Fluka, Buchs, Switzerland), reduced with 10 mM dithiothreitol (DuchefaBiochemie, Haarlem, the Netherlands) for 30 min, and alkylated with 10 mM iodoacetamide (Sigma-Aldrich, St. Louis, USA) for 30 min in the dark. To digest complex protein samples, 80 ng trypsin (Promega, Madison, USA) was added and the samples were incubated overnight at 37°C under static conditions. To stop the digestion, the samples were acidified with a final concentration of 0.1% trifluoroacetic acid (TFA, Sigma-Aldrich, St. Louis, USA) and subsequently purified using ZipTips (Millipore, Billerica, USA). For this purpose, the tips were stepwise equilibrated with 30 μL acetonitrile (ACN, Fluka, Buchs, Switzerland), 30 μL 80% ACN/0.1% TFA, 50% ACN/0.1% TFA, 30 μL 30% ACN/0.1% TFA and finally 30 μL 0.1% TFA. Peptides were bound to ZipTips by pipetting 10 times 10 μL of the sample. Impurities were removed by washing with 50 μL 0.1% TFA and finally peptides were eluted with 20 μL 50% ACN/0.1% TFA and 20 μL 80% ACN/0.1% TFA. The final eluates were concentrated using a vacuum centrifuge (Eppendorf, Hamburg, Germany) and stored at 4°C until further use.

Erythrocyte ghost samples were centrifuged at 17000g for 20 min at 4°C. The pellet was resuspended in 4% SDS/0.1 M HEPES and incubated for 30 min at 37°C with agitation 1000 rpm. Protein concentration was determined with the Lowry assay using BSA as protein standard [26 (link)]. Normalized samples were applied to 12.5% ExcelGels and run on a Multiphor II Electrophoresis System (Amersham Biosciences) at 600 V, 30 mA, and 30 W for 140 min. Proteins were visualized by staining the gel with Coomassie PhastGel Blue R-350. The whole sample lines were divided into 15 equal parts, excised by hand, and placed in protein LoBind Eppendorf microcentrifuge tubes (Eppendorf, Hamburg, Germany). Excised gel pieces were destained overnight and washed for 2 hours with 50% MeOH in water containing 5% acetic acid. Gel pieces were then completely dehydrated with 100% acetonitrile in a vacuum centrifuge (Eppendorf, Hamburg, Germany) for 5 min. Subsequently, samples were reduced with 10 mM dithiothreitol in 100 mM ammonium bicarbonate and alkylated with 100 mM iodoacetamide in 100 mM ammonium bicarbonate. Finally, proteins were digested overnight at 37°C using 0.6 μg of trypsin gold in 50 mM ammonium bicarbonate. Peptides were eluted in 50 mM ammonium bicarbonate with 0.1% formic acid, and the volume was reduced to 20 μL using a vacuum centrifuge as described above.

The RNA extraction of a single individual whitefly was carried out using the ARCTURUS PicoPure kit with modifications [32 (link)]. Extracted RNA was subjected to DNase treatment using the TURBO DNA free kit as described by the manufacturer (Ambion Life Technologies CA, USA). Subsequently, the RNA was concentrated using a vacuum centrifuge (Eppendorf, Germany) at 25°C for one hour. The pellet was resuspended in 18 μl of RNase free water and stored at—80 C waiting further analysis. Integrity of RNA was quantified by 2100 Bio-analyser (Aligent Technologies).

For determination of ovarian ubiquinone (UQ) concentrations, whole ovaries and livers were homogenized in 0.5 mL of homogenization buffer (0.25 m sucrose, 10 mm Hepes buffer pH 7.4, 1 mm EDTA) with ten passes of the Teflon pestle homogenizer (Wheaton Overhead Stirrer, Wheaton Instruments, Millville, NJ, USA). After the total volume was made up to 500 μL with the homogenization buffer, 5 μL of the homogenates was used to determine their total protein content using the Bradford Reagent (Bio-Rad Mississauga, ON, Canada). To extract UQ, whole ovarian homogenates were mixed with an equal volume of hexane/ethanol for 10 min by vortexing. After centrifugation at 9000 g for 10 min, the hexane layer was collected and evaporated to dryness using a vacuum centrifuge (Eppendorf, Mississauga, ON, Canada). The quinone residue was then dissolved in 100% ethanol and analyzed by HPLC with UV detection at 275 nm (Beckman System Gold, Beckman Coulter Inc, Brea, CA, USA). A reverse phase C18 column (25.0 × 0.46 cm, 5 μm, Hichrom, Berkshire, UK) was used with an isocratic elution at a flow rate of 1.8 mL min⁻¹. The mobile phase was methanol/ethanol (70:30 v/v). The concentrations of ubiquinones were estimated by comparison of the peak area with those of standard solutions of known concentration. Finally, quinone amount was normalized to protein content in the samples.