Fluorescence detector

Kimchi juice was prepared by blending all of the kimchi samples. Kimchi juice was diluted with 0.9% saline solution (HAPS DW-9, HUKO FS Co., Ltd., Seoul, Korea) and filtered. Initially, 1 mL of 2% potassium chromate was added and titrated against 0.02 N AgNO₃ until reaching a red-brown color. The capsaicinoid content of kimchi was analyzed using high-performance liquid chromatography (HPLC). The sample was diluted, filtered with filter paper, and evaluated using an HPLC analyzer (Agilent Technologies, Santa Clara, CA, USA) coupled with a fluorescence detector (Agilent Technologies). Excitation and emission wavelengths were 208 and 325 nm, respectively. Capsaicinoid contents were calculated from the peak sizes for capsaicin and dihydrocapsaicin chromatograms.

Size exclusion chromatography (SEC) was applied to determine the enzyme concentration in the dilute phase. A solution containing 1 µM fusion protein in 25 mM Tris, 20 mM NaCl pH 7.5 was incubated for 45 min at room temperature. The dilute and dense phase were separated by centrifugation at 8000 × g for 30 min. The supernatant was carefully removed and loaded on a Superdex 200 Increase 5/150 GL (Cytiva) assembled on an Agilent 1200 Series HPLC coupled to a fluorescence detector (Agilent). The elution profiles were monitored using the intrinsic tryptophan fluorescence of the fusion proteins. The amount of protein in the supernatant was compared to a reference 1 µM protein solution at high salt concentration (25 mM Tris, 500 mM NaCl, pH 7.5) in order to estimate the percentage of enzyme recruited in the dense phase.
The percentage of recruited protein into the nanoclusters of Dbp1-NOX was determined by following the same protocol, but the dense phase was removed by ultracentrifugation at 180,000 × g for 120 min at room temperature.

The AA production profile of CFS was analysed as described by Henderson et al. [35 ] by using Agilent 1100 high performance liquid chromatograph (HPLC) (Agilent Technologies, USA). Derivatisation of AA was performed by using o-phthalaldehyde (OPA) and 9- fluorenylmethyl chloroformate (FMOC). The derivatised AA were separated on a Zorbax Eclipse Plus C18 reverse phase column (4.6 mm × 150 mm, 3.5 μm) (Agilent Technologies, USA). The bound AA were eluted with 40 mmol/L sodium dihydrogen phosphate monohydrate (NaH₂PO₄·H₂O) adjusted to pH 7.8 and a methanol-acetonitrile-deionised water mixture (9:9:2) at a flow rate of 2 mL/min. The OPA, FMOC and NaH₂PO₄·H₂O were analytical grade while the methanol and acetonitrile were HPLC grade that purchased from Merck. The eluted derivatised AA were detected by a fluorescence detector (Agilent Technologies, USA) at the excitation/emission wavelengths of 340/450 nm for primary AA and 266/305 nm for secondary AA. The AA concentration was quantified by referring to the calibration curve constructed by using AA standard (Sigma Aldrich, USA). The production of AA was calculated by deducting the highest concentration of each AA with their respective initial concentration. All analyses were performed in triplicates.

eATP, eADP, eAMP and eADO in enzyme reaction solutions of nondistended and distended preparations were measured using a reverse-phased gradient Agilent Technologies 1200 liquid chromatography system equipped with a fluorescence detector (Agilent Technologies, Wilmington, DE, USA) as described previously [9 (link),34 (link),82 (link)]. Areas of HPLC peaks were calculated and calibrated to individual etheno-derivatized purine standards of eATP, eADP, eAMP and eADO.

Aspartic acid (Asp), glutamic acid (Glu), glycine (Gly), and γ-aminobutyric acid (GABA) were measured by high performance liquid chromatography with a fluorescence detector (HPLC–FLD) procedure. The HPLC system consisted of a microbore reverse-phase column (particle size 5 μm, 150 mm × 4.6 mm; Model Venusil AA, Bonna-Agela Technologies, Shanghai, China), an Agilent 1100 pump (Agilent Technologies, PaloAlto, CA, USA) and a fluorescence detector (Agilent Technologies, USA). The mobile phase (pH 6.8) consisted of 100 mM disodium hydrogen phosphate and 30% methanol; a 1 μL sample was derivated with 5 μL o-phthalaldehyde before being injected into the detection system.