The polyene antibiotics were extracted from the wet producer biomass by 96% (v/v) aqueous ethanol for 24 h. Ethanol extracts were applied on chromatographic plates of TLC Silica gel 60 F254 (Merck, Darmstadt, Germany). The chromatography of the extracts was carried out in a system of solvents: butanol:acetic acid:water (3:1:1). The spots formed on the chromatographic plates were compared with the control. Prepurified heptaene candidine and tetraene compounds synthesized by S. netropsis IMV Ac-5025 were used as controls. Purification of the compounds was performed through chromatographic separation on chromatographic plates, TLC Silica gel 60 F254 (Merck, Darmstadt, Germany) in a solution system for polyene antibiotics. The spots formed at the appropriate height of the plate were analyzed using a Spectrodensitometer (Sorbfill TLC View, Kyiv, Ukraine) with the UV filter, with a wavelength of 365 nm, using standard software and identified as corresponding to each of the fractions. The software responded to the height of the peak, which corresponded to candidine or tetraene compounds. The peak area indicated the amount of antibiotic and was supplemented with a control [36 (link),37 (link)].
Tlc silica gel 60 f254
Manufactured by Merck Group
Sourced in Germany, United States, India, Switzerland
TLC Silica gel 60 F254 is a thin-layer chromatography (TLC) plate consisting of a thin layer of silica gel with a fluorescent indicator. The silica gel acts as the stationary phase, and the fluorescent indicator F254 allows for the visualization of separated components under UV light.
Automatically generated - may contain errors
Lab products found in correlation
Silica gel 60, by Merck Group (7 mentions)
Silica gel, by Merck Group (7 mentions)
Ethyl acetate, by Xilong (6 mentions)
Silica gel 60n, by Kanto Chemical (4 mentions)
Avance 3, by Bruker (4 mentions)
Methanol, by Merck Group (4 mentions)
500 mhz spectrometer, by Bruker (4 mentions)
Jnm eca600, by JEOL (3 mentions)
Avance 3 hd 500 mhz spectrometer, by Bruker (3 mentions)
Tlc silica gel 60 rp 18 f254s, by Merck Group (3 mentions)
Eczr 500, by JEOL (3 mentions)
Esi tof ms spectrometer, by Waters Corporation (3 mentions)
Drx 400, by Bruker (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (3 mentions)
N hexane, by Xilong (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Hexane, by Merck Group (2 mentions)
Tetrahydrofuran, by Merck Group (2 mentions)
Jnm al400, by JEOL (2 mentions)
220 protocols using tlc silica gel 60 f254
1
Extraction and Purification of Polyene Antibiotics
2
Doxorubicin Deprotonation and Characterization
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Commercial doxorubicin hydrochloride (Dox.HCl) was deprotonated to obtain hydrophobic Dox (hDox). Dox.HCl was dissolved in a mixture of chloroform and methanol (3:2, v/v) and incubated overnight with triethylamine (TEA) at 1:3 molar ratio of Dox to TEA which resulted in deprotonation of the sugar amino group (Shuai et al., 2004 (link); Kim et al., 2008 (link); Liu et al., 2012 (link); Wei et al., 2015 (link); Zhang et al., 2016 (link)). After solvent evaporation, the deprotonated Dox (hDox) powder was collected and kept in the freezer. The quality of hDox was evaluated by 1HNMR by comparing the main structure with Dox.HCl. The state of hDox was qualitatively determined by thin layer chromatography (TLC). The samples were dissolved in THF and spotted on silica gel TLC plate (TLC silica gel 60 F254, Merk, Darmstadt, Germany) by microcapillary. The plates were developed in the mobile phase consisting of dichloromethane, methanol, formic acid, and deionized water (82:24:2:1, v/v) and were examined under UV light. Testing was performed at least in triplicate.
3
Bioactive Compound Profiling of A. sulphureus
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The extract of A. sulphureus MME12 was subjected to TLC and agar overlay bioautography (Valgas et al. 2007 ). About 10 μL of ethyl acetate extract was spotted on pre-coated TLC silica gel plates (TLC Silica gel 60 F254, Merk, Germany) and air dried. The spotted TLC plates were eluted in an elution system of hexane and ethyl acetate (1:1). The developed chromatogram was air dried and observed in a UV chamber at 254 nm and 365 nm for clearly resolved bands and the corresponding Rf (Retention factor) values were calculated. The chromatograms were subjected to agar overlay bioautography against E. coli and the plates were incubated at 37 ± 2 °C for 24 h and observed for inhibition zone. The experiment was repeated thrice.
4
Phytochemical Separation by Preparative TLC
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The extract was subjected to TLC for the separation of phytochemicals. The pre-coated silica plates (TLC Silica Gel 60 F254, Merk, India) were used for the separation. Analytical separation of extract containing 25 µg dissolved residue was carried out in various solvent systems differing in polarity. A preparative TLC plate of 200 mm × 200 mm was used to separate 350 µg dissolved residue in the best solvent system (20% acetone in ethyl acetate) identified from the separation on analytical plates. Two such plates were simultaneously developed in one chromatography chamber. At the end of separation, the solvent front was marked, and the Rf value of each visible band was calculated.
5
Characterization of Plant Extracts by TLC
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The obtained fractions and the crude extract were characterized by TLC, for which 20 mg of each fraction was dissolved in 1 mL ethanol and later deposited an aliquot on a chromatographic plate (Merk, TLC silica gel 60 F 254 ) in circles. On these plates, different elution media were tested: Hexane (Sigma Aldrich), Hexane:AcOEt (8:2 and 5:5), AcOEt, and AcOEt:Methanol (1:1). Once eluted, the plates were visualized with UV light at 254 and 365 nm.
6
SBE-HP Isolation and Analysis
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SBE-HP was obtained as a CH2Cl2 extract of S. bicolor, as described previously [30 (link)]. The isolated brownish-red paste was analysed by thin-layer chromatography (TLC) (Supelco, TLC Silica gel 60 F254, 1.05554.0001; Sigma-Aldrich) and LC-MS (Figure 2 A,B). For LC-MS analysis, 0.6 mL isopropanol, one scoop of 0.5 mm glass beads, and one scoop of 2 mm ZrO beads were added to the SBE-HP sample in a microcentrifuge vial. The mixture was homogenised in a Tissuelyzer II at 30 Hz for 5 min, vortexed, centrifuged at 21,100× g for 5 min, and transferred to an LC vial for analysis. Chromatography was performed on a Waters BEH C18 100 × 2.1 mm, a 1.7-micron column with Mobile Phase A: 60%acetonitrile 40%water 0.1% FA 10 mM ammonium formate and Mobile Phase B: 10% ACN 90% IPA, 0.1% FA 10 mM ammonium formate. The column temperature was maintained at 60 °C, and a 0.5 µL sample was injected for each run. MS data were acquired in positive and negative ion modes.
7
Vomilenine Extraction from R. serpentina Leaves
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Mature leaves (600 g) from glasshouse grown R. serpentina were harvested for vomilenine extraction. The leaves were submerged in ethyl acetate for 30 mins and evaporated. The alkaloids were extracted first by 1 M HCl and ethyl acetate. The aqueous phase was basified with NaOH to pH over 8, and subsequently extracted with ethyl acetate to afford total crude alkaloids. The crude alkaloids were then separated by TLC-silica gel 60 f254 (Sigma-Aldrich) with solvent ethyl acetate/methanol (9:1, v/v). TLC harvested vomilenine was identified by LC-MS/MS and NMR spectra.
8
Heterologous Expression and Biotransformation of Vomilenine Derivatives
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E. coli strains BL21DE3 containing VR or tDHVR in pET30b(+) vectors were inoculated in 2 mL LB medium overnight at 37 °C in a shaking incubator. The overnight cultures were used to inoculate 200 mL fresh LB medium with appropriate antibiotics, which were further grown at 37 °C in a shaking incubator until OD600 reached 0.6. A final concentration of 0.1 mM IPTG was then added to the cultures and induced overnight in a shaking incubator at 15 °C. The induced cells were collected and resuspended in 20 mL Tris-HCl (pH 7.5) supplemented with 10% (v/v) LB. For 1,2-dihydrovomilenine production, 50 μg vomilenine was added to cells expressing VR. For 19,20-dihydrovomilenine production, 50 μg vomilenine was added to cells expressing tDHVR. For 17-O-acetylnorajmaline production, 50 μg vomilenine was added to an equal mixture of cells expressing VR and tDHVR. The reactions took place in a shaking incubator at 15 °C for 24 h, then it was extracted with 20 mL ethyl acetate. The evaporated extract was reconstituted in methanol and separated by thin layer chromatography using TLC-silica gel 60 f254 (Sigma-Aldrich) with solvent ethyl acetate:methanol (9:1, v-v).
9
Synthesis of Polymer Nanocomposites
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Bisphenol AF (>98%), 4,4′-oxydianiline (>98%), and 1-(2-furyl)methylamine (99%) were purchased from TCI America (Portland, OR, USA). Paraformaldehyde (>95%), tetrahydrofuran (≥99.9%), and TLC Silica gel 60 F254 (100 glass plates, 2.5 cm × 7.5 cm) were obtained from Sigma Aldrich (Burlington, MA, USA). Chloroform and sodium hydroxide (NaOH) were received from Fisher Scientific Company (Waltham, MA, USA). Deuterated Chloroform (CDCl3) (99.8%) was purchased from Cambridge Isotope Laboratories., Inc. (Tewksbury, MA, USA). Glycidyl isobutyl POSS (EP0418) was purchased from Hybrid Plastics Inc. (Hattiesburg, MS, USA). Woven 3M Nextel 312 ceramic fabric (Style AF-40, 5 harness satin weave, 1800 denier, heat cleaned) was purchased from Bristal Metal Products, Inc. (Westlake, OH, USA). All chemicals were used as received.
10
TLC Analysis of Carbon Dots
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Carbon dots were applied as a 20 μL spot onto the bottom of the silica gel-coated aluminum TLC plate (TLC silica gel 60 F254, Sigma-Aldrich, Canada). The plate was developed in [ethyl acetate (60), methanol (30), deionized water (15), and formic acid (1)] (V/V). The fluorescence of the samples was detected via UV light at 366 nm with a GAMAG UV-light cabinet.
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