1260 infinity 2 chromatography system

High-performance liquid chromatography (HPLC) (20 (link)–22 ) was used to measure the catechin and theaflavin content of PDCe. HPLC was performed using a 1260 infinity II chromatography system from Agilent Technologies. PDCe was injected onto a SiO₂ column (250×4.6 mm), previously equilibrated with a solution composed of solvent A (acetonitrile) and solvent B (0.4% aqueous phosphoric acid, v/v). Compounds were eluted from the column using the following program: 7–15% A in 0–13 min, 15–20% A in 13–35 min, 20–50% A in 35–70 min, 50–80% A in 70–90 min, 80–87% A in 90–115 min. The flow-rate of the chromatographic mobile phase was set as 1.0 mL/min and the effluent was detected at 278 nm for acquiring chromatograms.

For each construct, the washed cells were distributed evenly into two 1.5 ml Eppendorf tubes and centrifuged at 2300 rcf for 4 min. The cell pellets were solubilized in 300 μl buffer composed of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA supplemented with either 5 mM (0.5% w/v) LMNG or 40 mM (2% w/v) β-C12M, and 5 mM PMSF while nutating for 1 h at 4 °C. Insoluble material was removed by ultracentrifugation at 105,000g for 20 min. One hundred microliters of supernatant containing solubilized enzyme was injected at 0.5 ml/min onto a Superose6 Increase 10/300 GL column equilibrated in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, and 0.01% (w/v) LMNG buffer. The column was attached to an Agilent 1260 Infinity II chromatography system equipped with a fluorescence detector and a temperature-controlled multisampler. Sample elution profiles monitored EGFP fluorescence (Ex 475 nm, Em 515 nm) with an instrument gain of 15 and data collection frequency of 9.3 Hz (1 s response time). A second 100 μl injection of the same sample was performed to ascertain stability following 48 h incubation on ice.