Beckman system gold

Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on a Beckman System Gold HPLC (Beckman Coulter, Fullerton, CA, USA) as previously described [41 (link),42 (link)] for the 22 C. horridus venom samples described above. In total, 50 $$\mathsf{\mu }$$ g of venom protein was injected onto a Jupiter C18 column (250 × 4.6 mm; Phenomenex, Torrence, CA, USA) using a solvent system of 0.1% trifluoroacetic acid (TFA) in water (solvent A) and 0.075% TFA in acetonitrile (solvent B). Following five minutes at 5% B, a 1% per minute linear gradient of A and B was run to 25% B; this was followed by a 0.25% per minute gradient from 25% to 65% B at a flow rate of 1 mL per minute. Column effluent was monitored at 220 and 280 nm.

In order to identify the antibiofilm components of ES products, SPE eluate was fractionated using a reverse phase (RP)-HPLC chromatography (Beckman System Gold, USA) coupled with a C18 column (250 mm × 4.6 mm; 5 μm) (Grace, USA) at a flow rate 0.3 mL/min, by using a gradient from 0 to 90% (v/v) acetonitrile (containing 0.1% (v/v) trifluoroacetic acid), during 70 min. The fractions were lyophilized and dissolved in 100 μL of TSB and tested for antibiofilm activity against S. aureus. The active fraction was then fractionated using a C4 column (250 mm × 4.6 mm; 5 μm) (Grace, USA) and tested under the same conditions. The purity of active fractions was checked by electrophoresis on 16.5% Tricine-SDS-PAGE gel using a Mini-Protean II electrophoresis cell (Bio-Rad, CA, USA). Protein bands were detected after staining with Coomassie Brilliant Blue R-250.

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.

Myocardial tissue of the left ventricular apex was extracted from all hearts and immediately preserved in liquid nitrogen. ADO production in the myocardium was detected using a high-performance liquid chromatography system (Beckman System Gold; Beckman Coulter, Beijing, China). ADO standard product was purchased from Sigma-Aldrich (St. Louis, MO, USA) (23 (link)).