Ods am column

The method reported by Xu and Chang [14 (link)] was employed for the determination of flavonoids in legumes samples. A Waters HPLC system (e2695 Separations Modulek, Milford, MA, USA) equipped with YMC-Pack ODS-AM column (250 mm × 4.6 mm) at 4 °C was used for the separation of various flavonoids present in samples. TFA (0.1%) and acetonitrile were used as mobile phase A and mobile phase B, respectively, for elution. The flow rate was set at 1 mL/min. The gradient of the solvent system followed by 90% of mobile phase A at 0 min, 70% at 20 min, 60% at 30 min, 50% at 50 and 55 min, 80% at 59 min, and 90% at 60 min.

The DNA encoding VSTx1 was inserted into the pET-30 Xa/LIC plasmid (Novagen) with a Factor Xa cleavage site between an N-terminal hexahistidine tag and VSTx1 sequences. VSTx1 was expressed in E. coli BL21 (DE3) cells. The uniformly ¹⁵N- and ¹³C, ¹⁵N-labeled samples were prepared by growing the transformants in M9 minimal medium containing ¹⁵NH₄Cl, glucose or [¹³C] glucose and [¹⁵N] or [¹³C, ¹⁵N] Celtone® base powder, respectively. Similarly, the uniformly ²H, ¹⁵N-labeled samples were prepared by growing the transformants in M9 minimal medium containing 99% ²H₂O, ¹⁵NH₄Cl, [²H] glucose and [²H, ¹⁵N] Celtone® base powder. The cells were grown at 37 °C to an A₆₀₀ of 0.6 to 0.8 and protein expression was induced with 1.0 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) for 6 h at 37 °C. The cells were harvested and lysed by sonication, followed by centrifugation. The pellets were solubilized by 8.0 M urea. VSTx1 was purified from the supernatant of the centrifuged lysate by a HIS-Select® Nickel Affinity Gel (Sigma) column. The eluted VSTx1 was refolded by dialysis, followed by digestion with Factor Xa protease (Novagen). The cleaved hexahistidine tags were removed by HIS-Select® Nickel Affinity Gel column. VSTx1 was further purified by the reverse phase high performance liquid chromatography using an ODS-AM column (YMC).

All the starting materials were obtained from G. L. Biochem. (Shanghai) or Sangon Biotech. (Shanghai). Commercially available reagents were used without further purification, unless noted otherwise. All other chemicals were reagent grade or better. PBS buffer (0.01 M, pH 7.4) was prepared with pills purchased from Sangon Biotech. Co., Ltd. (Shanghai, China). Ultrapure water (18.2 MΩ. cm) was used throughout the experiment. HepG2 human liver cancer cells were supplied by the Molecular Biology Laboratory of Anhui Medical University. High resolution ESI/MS spectra were obtained on a GCT premier mass spectrometer (Waters). HPLC analyses were performed on an Agilent 1200 HPLC system equipped with a G1322A pump and in-line diode array UV detector using a YMC-Pack ODS-AM column with CH₃OH (0.1% of TFA) and water (0.1% of TFA) as the eluent. ¹H-NMR spectra were obtained on a 300 MHz Bruker AV300. Cells were routinely cultured in Dul-becco's modified Eagle's medium (DMEM, Hycolon) supplemented with 10% fetal bovine serum at 37°C, 5% CO₂, and humid atmosphere.