Lc20 prominence hplc system

Natural compound containing C. vicina AMP complex was characterized by a combination of reversed phase HPLC, MS and bacterial growth inhibition assays. 1 mg of the lyophilized compound was dissolved in deionized water and applied to Shimadzu LC20 Prominence HPLC system equipped with analytical column C18 Vydac (4.6 х 250 mm, 5 μm, Grace), equilibrated with 0.05% TFA. The column was eluted with a linear gradient of acetonitrile (ACN) from 0 to 50% in acidified water (0.05% TFA) for 50 min [17 ]. Chromatographic fractions were automatically collected with 1 min intervals. The fractions’ optical densities were registered by means of a UV detector at two fixed wavelengths 214 and 280 nm. The fractions were lyophilized, dissolved in deionized water and tested against M. luteus A270 and E. coli D31 using the plate growth inhibition assay described below. Active antibacterial fractions were analyzed by MS (MicroTOF ESI, Bruker Daltonics) and experimentally determined masses were compared with the previously published characteristics of C. vicina individual AMPs [8 (link), 10 ]. The peptides were sequenced by Edman degradation method as described [10 ].

The complex AMPs were previously isolated and characterized by a combination of reversed phase HPLC, MS and Edman degradation methods [25 , 35 ]. In that work 1 mg of the lyophilized compound was dissolved in deionized water and applied to Shimadzu LC20 Prominence HPLC system equipped with analytical column C18 Vydac (4.6 × 250 mm, 5 μm, Grace), equilibrated with 0.05% TFA. The column was eluted with a linear gradient of acetonitrile (ACN) from 0 to 50% in acidified water (0.05% TFA) for 50 min. Chromatographic fractions were automatically collected with 1 min intervals. The fractions’ optical densities were registered by means of a UV detector at two fixed wavelengths 214 and 280 nm. The fractions were lyophilized, dissolved in deionized water and tested against planktonic M. luteus A270 and E. coli D31strains using the plate growth inhibition assay. Anti-biofilm activity of the fractions against one-day biofilms formed by E. coli ATCC 25922 и S. aureus 203 was analyzed by TTC and crystal violet assays as described below.