Analytical thin-layer chromatography (TLC) procedures Analytical TLC separations
were performed on Avicel® Microcrystalline Cellulose Uniplates (5 × 20 cm, 250 μm layer, glass-backed) ��-Nicotinamide in vitro and on Hard-Layer Silica Gel GHL Uniplates (5 × 20 cm, 250-μm layer, glass-backed, with an inorganic binder). For chromatography on cellulose plates, the solvent consisted of ethyl acetate:isopropanol:water (7.5:15:10). For chromatography on silica GHL plates, the solvent consisted of ethyl acetate:isopropanol:methanol:water (5:5:18:2). Unless otherwise indicated, the chromatographic samples (200 μL of the test solution) were applied to an origin line located 3 cm from one end of the plate as previously described [11]. The chromatograms
were developed over a distance of 12 cm from the origin. The developed chromatograms were dried and sprayed with a ninhydrin solution consisting of 0.25% (w/v) ninhydrin dissolved in 95% (v/v) ethanol containing 3.0 mL of glacial acetic acid per 100 mL of final solution. Color development was achieved by heating the sprayed chromatograms in an oven at 80-90°C for 15 min. The distribution of antimicrobial activity on the cellulose TLC chromatograms was determined in our standard agar diffusion assay. For this purpose, the chromatogram was divided into twelve 1-cm zones located between the origin and the solvent front. The cellulose from each zone (1 × 5 cm area) was scraped into separate 2.0-mL microfuge tubes, suspended in 1.33 mL of deionized water, and vortexed repeatedly to give a solution www.selleckchem.com/products/Cediranib.html representing a 3-fold concentration relative to the original culture filtrate. The cellulose was pelleted by centrifugation (10,000 rpm,
10 min, Sorvall MC 12V Minifuge), and the supernatant from each tube was filter sterilized (0. 2 μm Acrodisc 13 mm syringe filter) prior to testing in the agar diffusion assay. Sephadex G-15 column chromatography Sephadex G-15 (107 grams, medium grade) was swollen in deionized water and packed into a column (2.5 × 68 cm, 335 mL bed volume) in the same solvent. The column Isotretinoin was washed extensively with deionized water prior to initial sample loading and between column runs. Details of column fractionations are given in the legends to the corresponding figures. Chrome Azurol S assays of Sephadex G-15 column fractions Aliquots of Sephadex G-15 column fractions were assayed for phosphate (a major contaminant from the medium) and for amino acids using Fe-CAS and Cu-CAS reagents respectively. (The specificities of these reagents are illustrated in Additional files 5 and 6.) The reagents, prepared according to Shenker et al. [44], were composed of 210 μM CAS and 200 μM of either CuSO4 or FeSO4 in 40 mM MES buffer. The resulting solutions were adjusted to either pH 5.5 (Cu-CAS) or 5.7 (Fe-CAS) with NaOH.