Lipid analysis by two-dimensional thin-layer chromatography (2D-TLC) was done according to [10 (link)]. For calli lipids, 400 μg were loaded on silica plates 20 × 20 cm (Merck Millipore). For mitochondrial lipids, 150 μg of lipids were loaded on high-performance TLC (HPTLC) silica plates 10 × 10 cm (Merck Millipore). After separation, each lipid spot was analyzed by quantification of the fatty acid methyl esters (FAMEs) using gas chromatography (GC) according to [10 (link)].
Silica plates
Manufactured by Merck Group
Sourced in Germany
Silica plates are a type of laboratory equipment used for thin-layer chromatography (TLC). They consist of a thin layer of silica gel coated onto a rigid support, typically a glass, aluminum, or plastic plate. Silica plates provide a stationary phase for the separation and analysis of various chemical compounds.
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Lab products found in correlation
3h glycerol, by PerkinElmer (1 mentions)
Hamilton microsyringe, by Merck Group (1 mentions)
Amicon ultra 15 centrifugal filter, by Merck Group (1 mentions)
Acryloyl chloride, by Merck Group (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Ammonium persulfate ap, by Merck Group (1 mentions)
Tetramethylethylenediamine temed, by Merck Group (1 mentions)
Sephadex lh 20, by GE Healthcare (1 mentions)
Tween 80, by Merck Group (1 mentions)
Span 80, by Merck Group (1 mentions)
Automatic tlc sampler 4, by CAMAG (1 mentions)
Phosphate buffer tablets, by Merck Group (1 mentions)
Naproxen, by Merck Group (1 mentions)
Dimethylaminopyridine, by Merck Group (1 mentions)
Flurbiprofen, by Merck Group (1 mentions)
2 methoxyethyl acrylate, by Merck Group (1 mentions)
Triethylamine, by Merck Group (1 mentions)
Tbcl3 6h2o, by Merck Group (1 mentions)
Eu cf3so3 3, by Merck Group (1 mentions)
15 protocols using silica plates
1
Lipid Analysis by 2D-TLC and GC
2
Characterization of Fluorinated Organic Compounds
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All materials and solvents were purchased from Merck and Aldrich and were used without any additional purification. The melting points of the products were determined in open capillary tubes using BAMSTEAB Electrothermal apparatus model 9002. The 1H NMR spectra were recorded at 300 MHz. The 13C-NMR spectra were recorded at 75 MHz. The 19F-NMR spectra were recorded at 282 MHz. In the 19F-NMR spectra, up field shifts were quoted as negative and referenced to CFCl3. Mass spectra were taken by a Micro mass Platform II: EI mode (70 eV). Silica plates (Merck) were used for TLC analysis.
3
Hepatic Triglyceride Secretion Assay
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Analysis of hepatic triglyceride secretion was performed as previously described (18 (link)). Briefly, McA-RH 7777 cells were grown in MEM with 2% fetal bovine serum. After 24 hours, the medium was changed to MEM without fetal bovine serum plus 20 µM oleic acid, and after 48 h, DHT (10 nM) or vehicle was added for 24 hours. Cells were then incubated for 10 hours with [3H]glycerol (0.6 μCi/ml of culture medium; Perkin Elmer, Waltham, MA, USA) to trace triglycerides. Release of [3H]glycerol was chased in cold medium, and lipids were extracted in Folch solution (chloroform/methanol 2:1). Triglycerides were separated by thin layer chromatography on silica plates (Merck, Darmstadt, Germany) using a 2-phase system [first phase, chloroform:methanol:water (65:25:4 v/v); second phase, petroleumether:diethylether:acetic acid (80:20:1 v/v)], and [3H]glycerol was measured by scintillation counting (BeckMan Coulter, Fullerton, CA, USA).
4
Screening of PestE Variants by TLC
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For screening of PestE variants, 160 μL of 50 mm citrate buffer pH 5.0 and 40 μL enzyme solution (0.1 mg mL−1 final) were mixed. Then, 10 μL 200 mm HT in ethyl acetate (EtOAc) and 90 μL EtOAc were added. The reaction was incubated at 25 °C (1000 rpm) for 24 h. Reaction controls of 1 μL of the organic phase (OP) of each reaction were taken after 1, 2, 3, 4, 8, and 24 h and analyzed by TLC after 24 h to exclude that higher conversions were achieved at an earlier time point. TLC was performed using silica plates (Merck, Darmstadt, Germany) as solid and EtOAc as mobile phase. The staining was done with iodine (Rf(HT)=0.71; Rf(HTA)=0.91). After 24 hours, the reaction was stopped by adding 50 μL 2 m HCl. Subsequently, the reactions were extracted three times with 200 μL EtOAc each. The organic phase was dried over sodium sulfate, and analyzed by GC‐FID.
5
Metabolite Analysis by TLC in Microorganisms
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Strains were inoculated from GYM plates to GYM liquid broth and grown on a rotary shaker for 24-48 h at 28°C, 250 rpm. Fresh GYM broth was inoculated with 5% of these cultures and cultivation continued for next 10 days at 28°C, 250 rpm. Cells were separated by centrifugation for 10 min at 10 000 × g, 4°C, and cultivation broth was used for TLC analyses. Thirty μl of cultivation broth were applied on silica plates (Merck, Germany) by Hamilton micro syringe and the plates were placed in a chromatographic chamber pre-saturated for minimum 1 h with a mobile phase consisting of chloroform : methanol : ammonia (28%), 12:3:0.1. Detection of low-molecular weight substances was performed using a UV lamp at 254 and 366 nm, and by staining with coloring reagent. For this purpose developed plates were dried for 30 min at room temperature prior to spraying by orcinol reagent (orcinol 250 mg, ethanol 44 ml and sulphuric acid 6 ml). The plates were subsequently heated at 110°C for 2 min and spot appearance, position, and color were determined. Retention factor (Rf) values were determined by dividing distance moved by compound by distance moved by solvent.
6
Cleavage of Lipid-Linked Cell Wall Intermediates
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For reconstitution of the CapA1 mediated cleavage of the pyrophosphate-linkage of lipid-linked cell wall intermediates, purified CapA1 (8 µg) was incubated with 2 nmol lipid Icap, lipid IIIWTA, lipid IIPG in 10 mM MgCl2 and 50 mM Tris-HCl, pH 7.5. After incubation for 16 h at 30 °C, cleavage products were extracted from the reaction mixture with an equal volume of n-butanol/pyridine acetate, pH 4.2 (2:1, v/v) and analyzed by TLC on silica plates (Merck) according to Rick (chloroform, methanol, water, ammonium hydroxide, 88:48:10:1) and visualized by PMA staining75 (link).
7
Amino Acid Thin Layer Chromatography
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Samples of 2 μL of reaction mixtures were spotted on silica plates (Merck) with CSA, CA, glutamate, aspartate, and β-alanine as standards. The mobile phase was a mixture of butanol:acetic acid:H2O in the 3:1:1 ratio. After chromatography, amino acids were revealed upon treatment with 0.5% ninhydrin in acetone, followed by heating at 90 °C.
8
Purification and Characterization of Compounds
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All materials and solvents were purchased from Merck and Aldrich and were used without any additional purification. The melting points of the products were determined in open capillary tubes using BAMSTEAB Electrothermal apparatus model 9002. Mass spectra were taken by a Micro mass Platform II: EI mode (70 eV). Silica plates (Merck) were used for TLC analysis.
9
Analytical Characterization of Compounds
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All materials and solvents were purchased from Merck and Aldrich and were used without any additional purification. Mass spectra were taken by a Micro mass Platform II: EI mode (70 eV). Silica plates (Merck) were used for TLC analysis.
10
14C-Labeling of Cucumber Sugars
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The 14 C-labeling experiment of cucumber was done according to Ma et al. (2019) and Sui et al. (2017) . Briefly, when fruits (adjacent to leaf samples) at 9 DAA, the petiole of leaf samples was wrapped to prevent photosynthesis. Then, a sealable plastic bag was attached to those leaves and fed 14 CO 2 generated from NaH 14 CO 3 and excess 80% (v/v) lactic acid for 30 min under sunlight. The redundant 14 CO 2 was cleared by 3-M KOH. The leaves were exposed to ambient air for another 24 h after removal of the bag. Leaf, petiole, peduncle, and fruit MVB samples were put in liquid nitrogen, ground, and soluble sugar was extracted. Thin Layer Chromatography was used to separate the sugars on silica plates (Merck, Shanghai, China). The solvent system contained acetic acid, chloroform and water (7:6:1 in vol.). Maximum separation was achieved after running the plates 3 times. The radiolabeled spots were localized with X-ray film (Kodak Biome MR film, Rochester, USA). The spots were removed/cut out and placed in scintillation fluid. The percentage of each scraped radiolabeled sugar was calculated as the proportion of total sugars in the Ecoscint scintillation solution (National Diagnostics, Atlanta, GA, USA).
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