Hptlc alumina silica gel 60 plates

Inhibition assays with MraY_Cpn and the compounds were performed based on the activity assays for MraY_Cpn as previously described [32 (link)], with minor modifications, in a final volume of 50 µL. First, 2.5 nmol C₅₅-P (Larodan, Solna, Sweden) was dissolved in 0.43% (v/v) Triton X-100 by vortexing for 1 min. Then, 75 mM Tris-HCl (pH 7.5), 6 mM MgCl₂, 10% (v/v) DMSO, and UDP-MurNAc-pentapeptide obtained from Bacillus cereus DSM2302 (based on [93 (link)]) were added. Compounds were applied at the indicated concentrations and incubated with 2 µg MraY_Cpn at 30 °C for 90 min. The reaction products were extracted with 50 µL n-butanol-pyridine acetate (2:1, pH 4.2) by vortexing for 1 min. After centrifugation at 17,000× g for 5 min, components of the organic phase were separated by thin layer chromatography (TLC) on HPTLC alumina silica gel 60 plates (Merck) using chloroform–methanol–water–ammonia (88:48:10:1) as a mobile phase [94 (link)]. The TLC plates were stained with phosphomolybdic acid stain (2.5% (w/v) phosphomolybdic acid, 1% (w/v) ceric-sulphate, 6% (v/v) sulfuric acid) and visualized at 120 °C. The experiments were performed in duplicate.

Phosphatase assays were carried out in a final volume of 50 µl containing 10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 0.5 mM CaCl₂, 40 µM lipid II, 0.95 µM protein (protein content in liposomes), 1 µM LpoB(sol) and 2 nmol lipid II. The mixture was incubated for 4 h at 37 °C. Reactions were terminated by adding 50 µl n-butanol/pyridine acetate pH 4.2 (2:1). Samples were vortexed for 1 min and centrifuged for 3 min at 17,000g on a bench top centrifuge. The reaction products present in the organic phase were analysed by TLC on HPTLC alumina silica gel 60 plates (Merck KgaA) using chloroform-methanol-water-ammonia (88:48:10:1) as the mobile phase (Rick et al., 1998 (link)). TLC bands were stained with iodine and quantified using ImageJ software, the data were analysed using Excel.