Liquid scintillation mixture

The following isocratic RP-HPLC system was based on the methodology employed by Frolik et al. for the study of retinoids (Frolik et al., 1978 (link)). However, the system proved to be also efficient to separate different polyprenols such as POH and GGOH. The system was performed at 25°C using acetonitrile: 1% ammonium acetate (4:1) at a flow rate of 2 ml/min. It was employed a Hichrom^® Nucleosil^® 100 C18 column (250 mm × 4.6 mm) (Leicestershire, UK), a diode array detector (DAD) 170, and a fraction collector FC203B purchased from Gilson^® (Villiers-le Bel, France). The software used for data processing was the Trilution™ LC 3.0 System Software^®. Polyprenol’s elution was monitored at 220 and 350 nm and fractions were collected every 0.5 min. RP-HPLC fractions were dried at 50°C and then suspended in 0.5 ml of liquid scintillation mixture (PerkinElmer Life Sciences^®, MA, United States). The radioactivity of each fraction was monitored with a Beckman LS 5000 TD β-counter scintillation counter (Beckman^®, CA, United States). Metabolic profiles were analyzed by OriginPro 8.1^® software (OriginLab Corporation^®, MA, United States).

The assessment of radiolabeled proteins was performed following a similar protocol as described elsewhere [63 (link)]. Radiolabeled parasites were suspended in 100 μL of lysis buffer (2% w/v SDS, 60 mM DTT in 40 mM Tris-Base pH 9). The samples were then cooled at room temperature, and proteins were precipitated by adding 20% trichloroacetic acid (TCA) in acetone at 4°C. The samples were kept on ice for 5 minutes, and the proteins were collected by centrifugation at 12,000 × g for 10 minutes. The precipitate was washed three times with 80% acetone. Subsequently, the proteins were dissolved by incubating them at 90°C in alkaline buffer (0.5 M NaOH, 25 mM EDTA, 0.1 w/v SDS in water) for 30 minutes. Finally, 1 mL of liquid scintillation mixture (PerkinElmer Life Sciences, MA, USA) was added to the samples. After vortex, the radioactivity of samples was measured using a Beckman LS 5000 TD β-counter apparatus (Beckman, CA, USA) and results were analysed using GraphPad Prism software.