Jupiter c18 column

Venom samples were collected from H. waigiensis using a square-wave stimulator (Arthur H. T. Thomas Co. Scientific Apparatus, Philadelphia, PA, USA) to electrostimulate the venom gland at 25V DC, 0.5 ms pulse duration, at a frequency of 1 pulse/sec. The samples were pooled into 20 µL of MilliQ water and stored at −20 °C. Pooled samples collected on two separate dates were further pooled prior to analysis. A further five scorpions (two male and three female) were milked and the samples were stored separately in 30 µL of MilliQ water for LC/MS analysis.
Crude pooled venom was fractionated by reversed-phase HPLC (RP-HPLC) using a Phenomenex Jupiter^® C₁₈ column (250 × 10 mm, 10 µm, 100 Å; Phenomenex, Torrence, CA, USA). Fractionation of the venom components was achieved using a linear gradient of two mobile phases: H₂O/0.05% trifluoroacetic acid (TFA; Auspep, Tullamarine, VIC, Australia) (solvent A) and 90% acetonitrile (ACN; Sigma-Aldrich, St. Louis, MO, USA)/H₂O/0.045% TFA (solvent B). Separation used a gradient of 0–60% solvent B in 120 min, 60–90% solvent B in 5 min, 90% solvent B for 10 min, and 90–0% solvent B in 5 min, at a flow rate of 3 mL/min. The venom component elution was monitored at 214 nm and 280 nm, and 0.5 min fractions were collected.

Concentration of sCT in aerosolization tests was determined by HPLC system (Shimadzu LC-20AT liquid chromatograph, Shimadzu Co., Ltd., Kyoto, Japan) equipped with a reverse-phase Phenomenex Jupiter C₁₈ column (5 μm, 4.6 × 250 mm, Phenomenex Inc., Torrance, CA) at 40 °C with UV detection at 220 nm. Acetonitrile (mobile phase A)–0.02 mol/L TMAH solution (pH 2.5, mobile phase B) was adopted as mobile phase at 1.000 L/min. A program of dual gradient elution was run as T (min)/%mobile phase A: 0–15 min, 72%; 15–17 min, 63%; 17–30 min, 72%. Injection volume was adjusted to 100 μL. A calibration curve was constructed by sCT standard solutions of 0.8–50 μg/mL (R²=0.9997). Each sample was quantified in triplicate.

Structural alterations of NFAP and its mutants were examined by RP-HPLC, using an Agilent 1100 Series liquid chromatograph (Agilent technologies, Little Falls, DE, USA) and a Phenomenex Jupiter C18 column (250 × 4.6 mm; 10 μm particle size; 300 Å pore size; Phenomenex, Torrance, CA, USA). Linear gradient elution was carried out with 0.1% TFA in water (eluent A) and 80% acetonitrile and 0.1% TFA is water (eluent B) from 15% to 40% (B) over 25 min at a flow rate of 1.0 ml/min.

OsCEP1a (DVRHypTNPGHSHypGIGH), OsCEP1b (DVRHypTNHypGHSHypGIGH), OsCEP1c (GVRHypTNPGHSHypGIGH), and OsCEP1d (GVRHypTNHypGHSHypGIGH) were synthesized on a CS 136X synthesizer (CS Bio, Menlo Park, CA, USA) using Fmoc solid phase peptide synthesis chemistry. Hydroxyproline (Hyp) was introduced with Fmoc-Hyp(Boc)-OH purchased from Watanabe Chemical Industries, Ltd. (Hiroshima, Japan). The obtained crude peptides were purified by reverse-phase HPLC on Jasco, Inc. (Tokyo, Japan) preparative instruments with a Jupiter C18 column (5 µm, 300 Å pore size, 21.2-mm internal diameter ×250 mm) (Phenomenex, Torrance, CA, USA), and lyophilized to yield pure peptides.

A 600 mg sample of dried N. naja venom was dissolved in 0.1 M ammonium acetate buffer, pH 6.2, and applied to a Sephadex G50s column (4.5 × 150 cm) equilibrated in the same buffer. The column was eluted at flow rate 32 mL/min. The fractions obtained were pooled as shown in Figure S1A. Fraction V was further separated on a HEMA BIO 1000 CM column (8 × 250 mm) (Tessek, Prague, Czech Republic) in an ammonium acetate gradient from 5 to 700 mM (pH 7.5) in 140 min at flow rate 1.0 mL/min (Figure S1B). Fractions 14–18 were freeze-dried and further purified by reversed-phase chromatography on a Jupiter C18 column (10 × 250 mm, Phenomenex, Torrance, CA, USA) in a gradient of acetonitrile 25–35% in 60 min in the presence of 0.1% trifluoroacetic acid, at a flow rate of 2.0 mL/min. After freeze-drying, the obtained proteins were used for further studies. Molecular masses of the isolated products were determined by mass spectrometry [35 (link)]. A similar procedure was used to isolate cytotoxins from N. haje venom.