Reverse phase hplc

Catalytic activity of RDH11 was assayed in 25 mmol/L phosphate buffer saline (PBS), at 37 °C (reaction buffer) in siliconized glass tubes. The reductive activity of RDH11 toward all- trans-retinaldehyde substrate was analyzed using all- trans-retinol and all- trans-retinaldehyde (Roch). The stock solution of all- trans-retinaldehyde substrate was dissolved in DMSO. The 500 μl reactions were started by the addition of cofactor 0.3 mmol/L NADPH-Na4 and carried out for 15 min at 37 °C with 200 rpm shaking. The amount of cell lysates in the reaction mixture is around 250 μg. The reactions were terminated by the addition of an equal volume of cold methanol: n-butyl alcohol 95:5 supplemented with 100 μg/ml butylated hydroxytoluene. Retinoids were analyzed using a Waters Alliance HPLC system. Elution was monitored at 325 nm. Ten-microliter aliquots were analyzed by reverse-phase HPLC (SHIMADZU, Japan) using a C18 column (4.6 × 150 mm), and the mobile phase consisted of acetonitrile: 30 mmol/L ammonium acetate (85, 15, v/v). The flow rate was 1.5 ml/min. Under these conditions, all- trans-retinol and all-trans-retinaldehyde eluted at around 11.27 and 12.64 min, respectively. The reaction products were quantified by summing the areas of both peaks of retinol and retinal. The ratios of the produced retinol were analyzed.

Ficoll-Paque Plus was sourced from GE Healthcare Japan Corporation (Tokyo, Japan). EasySep™ Human Basophil Enrichment Kit was sourced from STEMCELL Technologies (Vancouver, Canada). Reverse-phase HPLC was sourced from Shimadzu (Kyoto, Japan). Tetramethylrodamin B isothiocyanate (TRITC)-phalloidin and adenosine were sourced from Sigma-Aldrich Japan (Tokyo, Japan). Human IgE Quantitative ELISA kit was sourced from Bethyl Laboratories, Inc., (Montgomery, TX, USA). Human IgE Purified (AG30P) was sourced from MILLIPORE (Burlington, MA, USA). Human IgE (P50) was sourced from Nordic-MUbio (Susteren, the Netherlands). Human IgE (HE1) was sourced from Bioporto Diagnostics A/S (Hellerup, Denmark). Fluorescein Labeling Kit-NH2 was sourced from Dojindo laboratories (Kumamoto, Japan). Anti-IgE receptor-FITC, anti-CD123-PE, anti-CD203c-APC, anti-IgE-APC, and anti-CD203c-FITC were sourced from BioLegend (San Diego, CA, USA). Allergenicity^® Kit was sourced from Beckmann Coulter (Brea, CA, USA). IL-3 was sourced from R&D Systems Inc (Minneapolis, MN, USA).

The apparatus used for the analysis was a reverse phase HPLC (Shimadzu, Kyoto, Japan) equipped with a fluorescence detector. The analytical column used was a Symmetry® C-18 3.9x150 mm with 5 μm particle size from Waters (Massachusetts) and the guard column was Silfilter STD C-18 3.0x10 mm. The column was maintained at 40°C. Analysis was run at a flow rate of 1 ml/min by an isocratic mobile phase using a mixture of acetonitrile/methanol/water (15/15/70 v/v/v). The total run time was 20 min. An aliquot of a 10 μl sample extract was injected into the chromatographic system and detection was carried out by a fluorescence detector (excitation and emission wavelengths were 360 and 440 nm, respectively). Chromatograms were displayed with Class VP LC software.

Lyophilized Cvv venom (10 mg) was dissolved in 1 ml of 20 mM Tris–HCl, 150 mM NaCl, pH 8.8 and centrifuged at 5,000 g for 2 min. The supernatant was applied onto a reverse phase analytical C₈ column (5 µm, 250×4.6 mm) (Kromasil, Sweeden), previously equilibrated with the same buffer. Venom proteins were separated by reverse phase HPLC (Shimadzu, Japan). Fractions (0.7 ml/tube) were collected at a 1 ml/h flowrate. A linear gradient of water/acetonitrile containing 0.1% trifluoroacetic acid (TFA) was used. The elution profile was monitored by absorption at 280 nm, and the molecular homogeneity of the relevant fractions was verified by SDS-PAGE. Fractions containing protein peaks were dried in a Speed-Vac (Savant, Thermo Scientific, USA) and resuspended in distilled water prior to protein quantification by the Bradford method. Molecular mass determination was performed by MALDI-TOF and by electrospray ionization (ESI) mass spectrometry using a Voyager-DE Pro and a QTrap 2000 (both from Applied Biosystems), respectively.

The fat- and water-soluble vitamins in the various samples were analyzed following the methods described by Maiyo et al. [31 (link)] and Bhatnagar-Panwar et al. [32 (link)]. Liquid chromatography mass spectroscopy (LC-MS) coupled with a diode array detector (HPLC-30AC; Shimadzu, Tokyo, Japan) and a C18 column (100 × 3.00 mm, 2.6 µm polar; Phenomenex, Torrance, CA, USA) at 30 °C, was used and each sample and standards were analyzed for 12 min at a flow rate of 0.4 mL/min and injection volume of 10 µL. The number of water-soluble vitamins in the sample were calculated by comparing peak area of samples to peak area of the standards.
For the fat-soluble vitamins, analyses were performed using reverse-phase HPLC (Shimadzu, Tokyo, Japan) linked to a SPD-M2A detector. The injection volume of each sample was 10 µL with a total flow rate of 0.4 mL/min for 10 min. Standards for retinol and α- and γ-tocopherol were prepared at four different concentrations each and used for the calibration curve. Peaks were identified by their retention time and the absorption spectra were compared to the standardized spectra. Vitamin concentrations were calculated by comparing the peak area of samples to the peak area of the standardized spectra.