5735 silica gel 60f254

Relebactam (2 mg/mL) was added to recombinant Bla_Mab (0.01 mg/mL) and incubated for 5 min at room temperature, before addition of Penicillin V (4 mg/mL) for a further 10 min incubation at room temperature. Alongside appropriate control reactions (Fig. 2a), 1 µL of the reaction was spotted onto aluminium backed silica gel plates (5735 silica gel 60 F₂₅₄, Merck) and dried before being subjected to Thin Layer Chromatography (TLC) using ethyl acetate:water:acetic acid (C₄H₈O₂:H₂O:CH₃COOH) (3:1:1, v/v/v). Once dry, plates were visualised by being dipped into KMnO₄ TLC stain with light charring.

M. marinum strains were grown to mid-log growth phase in Sauton's medium containing or not magnesium sulfate and labeled with 0.5 µCi/ml [1-¹⁴C]propionate (specific activity of 54 mCi/mmol, American Radiolabeled Chemicals, Inc) for 24 h. The non-polar fraction was separated from the polar fraction as described [27] . The [1-¹⁴C]-propionate-labeled apolar lipids were extracted by adding 2 ml of CH₃OH/0.3% NaCl (100/10; v/v) and 2 ml of petroleum ether to the cell pellet followed by stirring for 1 h. After centrifugation, the upper petroleum ether layer was removed and 2 ml of petroleum ether was added to the lower phase. The combined petroleum ether extracts were then evaporated under a nitrogen stream to yield apolar lipids that were resuspended in CH₂Cl₂ and analyzed by TLC using silica gel plates (5735 silica gel 60F254; Merck, Darmstadt, Germany). Equivalent amounts of lipids (20 000 cpm) were spotted on TLC plates that were run in various solvent systems: Petroleum ether/diethylether 9∶1 (v/v) for PDIM; chloroform/methanol 19∶1 (v/v) for TDM and PGL and chloroform/methanol 99∶1 (v/v) for PAT. TLCs were exposed to a Kodak Biomax MR film for 24 h.

De novo fatty acid and mycolic acid biosyntheses were followed by labeling 5 ml culture aliquots with 1 µCi/ml [1-¹⁴C]-acetate (specific activity: 55.3 mCi/mmol; Perkin Elmer) for 1 h at 37°C. Fatty acid and mycolic acid methyl esters were extracted from samples containing equivalent amounts of bacteria as described by [32] (link). The resulting solution of FAMEs and MAMEs was assayed for radioactivity in a Beckman liquid scintillation counter and then subjected to TLC using silica gel plates (5735 silica gel 60F254; Merck). Samples were normalized by culture OD or total cpm and developed in hexane∶ethyl acetate (9∶1, v/v). Autoradiograms were produced by overnight exposure to Kodak X-Omat AR film to reveal ¹⁴C-labelled FAMEs and MAMEs.