Lc ms instrument

Crude antibacterial compound was extracted from the cell-free culture broth of strain JAJ13 using an equal volume of ethyl acetate. The antibacterial compound was partially purified using silica column chromatography with gradient solvent system (hexane: ethyl acetate). Fractions were analyzed on TLC and tested for antibacterial activity by disc diffusion method as described in a following section. A fraction showed antibacterial activity was analyzed on HPLC instrument (Shimadzu, Japan), using shim-pack CLC ODS (4.6 × 15 mm) column, and a 350 nm detector. Methanol and water (65:35, v/v) were adopted as a mobile phase with a flow rate of 1.0 ml/min at 25°C. Filtered sample was injected into the column and the relative retention time was recorded.
The partially purified antibiotic was further analyzed using a LC-MS instrument (Waters, Germany) consisting of Alliance separations module e2695; ACQUITY QDa detector, and a C18 reversed-phase column. Solvent A consisted of 0.01% (v/v) formic acid in water. Solvent B consisted of 0.01% (v/v) formic acid in acetonitrile. The mass spectrometer was operated in the positive-full-scan (m/z 150–700) mode.

All ¹H-NMR spectra are reported in parts per million (ppm) and were measured relative to the signals of DMSO (2.5 ppm). ¹³C-NMR spectra are reported in ppm relative to those of residual DMSO (40 ppm). The mass spectra of the chemicals were obtained using a Waters LC–MS instrument equipped with a single-quadrupole mass detector and an electrospray ionization source (Waters ACQUITY QDa), while those of the proteins were obtained using Agilent 6100 equipment with a series of triple-quadrupole mass spectrometers (Agilent Technologies, CA, USA). Protein purification was performed using AKTA UPC 900 FPLC system (GE Healthcare), and absorption spectra were obtained at room temperature using a UV–visible spectrometer (Agilent 8453, Agilent Technologies). Fluorescence spectra were obtained using a microplate reader equipped with SkanIt 2.4.3 RE software for Varioskan Flash (Varioskan Flash, Thermo Fisher Scientific Inc.). Cell fluorescent images were recorded at room temperature by confocal and fluorescence microscopy (FV1000, OLYMPUS).