AGE and SAC were analyzed using a CAMAG HPTLC comprising of applicator- linomat5, digistore-2, multi-wavelength scanner, transparent chromatographic tank, HPTLC pre-coated silica plate; silica gel 60 F254, 10 × 10 cm (Merck). Injector and detector temperatures were set at 60 °C. Mobile phase used was ethyl acetate: acetic acid: water: acetone (9:1:1:1) and ninhydrin detector was used. A volume of 20 μL of standard and sample solutions was injected, the area due to SAC in the chromatogram was identified and the amount of SAC in AGE sample was calculated.
Hptlc
Manufactured by Merck Group
Sourced in Germany
HPTLC (High-Performance Thin-Layer Chromatography) is a laboratory analytical technique used for the separation, identification, and quantification of various chemical compounds. It is a versatile method that allows for the rapid and efficient analysis of complex mixtures. HPTLC utilizes a thin layer of adsorbent material, typically silica gel, coated on a glass, metal, or plastic plate, as the stationary phase. Samples are applied to the plate, and the compounds are separated based on their differing affinities for the stationary phase and the mobile phase (a solvent or a solvent mixture) as it travels up the plate.
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Lab products found in correlation
Silica gel 60 f254, by Merck Group (2 mentions)
Tlc visualizer documentation system, by CAMAG (1 mentions)
96 well filter plate, by Merck Group (1 mentions)
Oasis hlb 1cc extraction cartridges, by Waters Corporation (1 mentions)
Tunicamycin, by Fujifilm (1 mentions)
Fla 7000 analyzer, by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Millex lg, by Merck Group (1 mentions)
13 protocols using hptlc
1
Quantitative Analysis of Amino Acids in Herbal Extract
2
Lipid Extraction and Chromatographic Analysis
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Lipid extraction and chromatography were performed mostly as previously described [62 (link)].
Cells in culture were washed with phosphate-buffered saline (PBS) and harvested from the dishes with a cell scraper. After centrifugation, the pellets of cells were measured for the wet weight and were pooled for lipid extraction. Lipids fromT98G control (433 mg of wet weight) and GOLPH3 KD (371 mg of wet weight) were extracted with chloroform:methanol (2:1, v/v) at 4 °C for 24 h and centrifuged. The supernanatant was collected and the pellet subjected to a second lipid extraction by adding chloroform:methanol:water (30:60:8) at 4 °C for 24 h and centrifuged. Both supernanatants were mixed and subjected to two Folch partitions, first by adding 0.2 vol. of water and then 0.2 vol. of methanol:water (1:1). The resulting upper phases were freed from water-soluble contaminants by being passed through a Sephadex G-18 column. The lipid extract was used for thin-layer chromatography (TLC) analysis, supplemented with the appropriate amounts of standard gangliosides, and chromatographed on high performance TLC plates (HPTLC; Merck) using C:M:0.2% CaCl2 (60:36:8 v/v) as solvent. The bands were visualized by resorcinol-HCl spray and heating at 100 °C for 10 min.
Cells in culture were washed with phosphate-buffered saline (PBS) and harvested from the dishes with a cell scraper. After centrifugation, the pellets of cells were measured for the wet weight and were pooled for lipid extraction. Lipids fromT98G control (433 mg of wet weight) and GOLPH3 KD (371 mg of wet weight) were extracted with chloroform:methanol (2:1, v/v) at 4 °C for 24 h and centrifuged. The supernanatant was collected and the pellet subjected to a second lipid extraction by adding chloroform:methanol:water (30:60:8) at 4 °C for 24 h and centrifuged. Both supernanatants were mixed and subjected to two Folch partitions, first by adding 0.2 vol. of water and then 0.2 vol. of methanol:water (1:1). The resulting upper phases were freed from water-soluble contaminants by being passed through a Sephadex G-18 column. The lipid extract was used for thin-layer chromatography (TLC) analysis, supplemented with the appropriate amounts of standard gangliosides, and chromatographed on high performance TLC plates (HPTLC; Merck) using C:M:0.2% CaCl2 (60:36:8 v/v) as solvent. The bands were visualized by resorcinol-HCl spray and heating at 100 °C for 10 min.
3
HPTLC Analysis of Methanolic Extracts
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The methanolic extract and fractions of FF were analyzed by high performance thin layer chromatography (HPTLC; Merck, Darmstadt, Germany). Samples were spotted on HPTLC plates and developed using chloroform: methanol (50: 1 (v/v)). After drying, plates sprayed with a chromogenic reagent (acetic acid: sulfuric acid: p-anisaldehyde = 100: 2: 1) and scanned using a TLC visualizer documentation system (CAMAG, Switzerland) using white light.
4
Microsomal Membrane-Based Ceramide Synthase Assay
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Microsomal material was prepared from MSY23-3C pESC-LEU_AtIPCS2 and MSY23-3C pESC-LEU_AUR1 as previously described33 (link). IPCS turnover was assayed using HPTLC (Merck) and imaged using a Fuji FLA−3000 reader and AIDA Image Analyser® software (version 3.52) as previously described17 (link). Subsequently, a 96-well plate assay was formatted based on the protocol described by Mina et al.17 (link). Following optimisation of substrate concentration and incubation time, each compound at the desired concentration (100 µM to 46 nM; in triplicate), was incubated in 96-well plates (Corning® Costar®) in phosphate buffer (71.4 mM, pH 7.0) with soybean PI (100 µM, final concentration, Avanti), NBD-C6-phytoceramide (15 µM; ThermoFisher) and microsomal membranes (0.3–0.4 Units17 (link)). Following incubation for 60 minutes (or 40 minutes for MSY23-3C pESC-LEU_AUR1 membranes) at 30 °C the reaction was quenched by the addition of 200 µl methanol per well, the reaction product separated using exchange chromatography in 96-well filter plates (Millipore)17 (link) and the fluorescence measured at Ex460/Em540 using a fluorescence Microplate Reader (Biotek Synergy H4 with Gen5™). Analyses were carried out using GraphPad Prism 7.
5
Cerebellum Lipid Extraction and Analysis
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Cerebellum from 6-week-old mice was homogenized with four volumes of ice-cold water in an all-glass Potter-Elvehjem homogenizer; 250 μl of homogenate (50 mg of wet tissue) was extracted by addition of 1.2 ml of methanol and 2 ml of chloroform. After incubation of the samples at 37 °C for 1 h, 1 ml of methanol was added, and the extracts were centrifuged at 2,000 g for 10 min. The pellet was re-extracted with 2 ml of chloroform/methanol/water (1/2/0.8, v/v/v) at 37 °C for 2 h. The combined supernatants were concentrated by Speed-Vac, and the dried samples were dissolved in 2 ml of methanol and saponified. After neutralization, samples were diluted with 2 ml of water and desalted using OASIS HLB 1 cc extraction cartridges (Waters). Thin-layer chromatography was performed using HPTLC (Merck) and developed with chloroform/methanol/0.02% CaCl2 (5:4:1, v/v). After staining with orcinol–sulfuric acid, GSLs were identified by comparing their RF to those of authentic GSL standards.
6
Radiolabeling and Analysis of GPI Biosynthesis
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Approximately 2 × 106 of CHO-PIGS KO, HEK293-PIGS KO, PIGS-UBE2J1 DKO and PIGS-SYVN1 DKO cells were precultured in normal medium overnight, cells were washed with wash medium (glucose-free DMEM buffered with 20 mM HEPES) and incubated for 1 h at 37 °C in 1 mL of reaction medium (glucose-free DMEM buffered with 20 mM HEPES, pH 7.4, and supplemented with 10% dialyzed FBS (Gibco), 10 μg/mL tunicamycin (Wako), and 100 μg/mL glucose). [2-3H] Mannose (American Radiolabeled Chemicals) was added to 25 Ci/ml, and the cells were incubated for 1 h at 37 °C in 5% CO2. After labeling, the cells were washed twice with 1 ml of PBS and released from culture plates by cell scraper. Cells were pelleted and washed with 1 ml of cold PBS. Radiolabeled GPIs were extracted by 1-butanol and separated by HPTLC (Merck)74 (link), and visualized by a FLA 7000 analyzer (Fujifilm). Quantitative analysis was performed by JustTLC (SWEDAY).
7
Isolation and Purification of Lipid Extracts
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H. pylori was washed with PBS and then treated with chloroform/methanol (2:1, vol/vol) for 1 d. This mixture was partitioned by centrifugation and the lower organic phase was used as the lipid extract. To isolate αCAG or αCPG, the lipid extracts were separated by HPTLC (Merck) and visualized by copper(II) acetate-phosphoric acid (180°C, 15 min) or orcinol (120°C, 5–15 min) staining. The fractions containing these lipids were scraped from the plate, and chloroform/methanol (2:1, vol/vol) was used to elute the lipids. The purified lipids were filtered using Millex-LG (0.2 μm; Merck) to remove silica gel contamination.
8
Analytical Instrumentation for Characterization
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The instruments that were used included a densitometer with TLC scanner 3 operated by Wincats (version 1.4.3) planar chromatograph software as data manager and integrator, Linomat 5, a semi automatic applicator with Hamilton syringe of 100 μL capacity for sample application and CAMAG developing tank single rectangular with internal dimensions (21.6 × 11.2 × 6) cm all were made from CAMAG, Muntez, Switzerland. The TLC (5 × 10) cm, HPTLC (10 × 10) and (20 × 10) cm plate pre coated with silica gel 60 F 254 were made from Merck, Darmstadt, Germany and a fluid bed drier that was made from GEA Niro pharma system, Bubendorf, Switzerland. A tablet press, EKO1 made in Germany and turbular mixer made in Switzerland.
9
Lipid composition analysis by HPTLC
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Aliquots of 30 µg from TL extracts were used, and LC was separated by high-performance thin-layer chromatography (HPTLC, Merck KGaA, Darmstadt, Germany) in a single-dimensional double-development. 2-propanol/chloroform/methylacetate/methanol/0.25% KCl (5:5:5:2:1.8, v/v) and hexane/diethyl ether/acetic acid (20:5:0.5, v/v) were the solvents for polar lipid and neutral fractions, respectively. LC was visualized by charring at 160 °C after spraying with 3% (w/v) aqueous cupric acetate containing 8% (v/v) phosphoric acid and quantified in area % in an HPTLC Visualizer (CAMAG, Muttenz, Switzerland) as described by Olsen and Henderson [45 (link)].
10
Isolation and Characterization of Inositol Phosphoceramides
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Lipids were extracted from ~2 × 108 promastigotes isolated from either log phase or 48 h after stationary phase by homogenization with isopropanol/hexane/water (IHW) (55:20:25, v/v/v) and with chloroform/methanol (CM) (2:1, v/v) (Silveira et al., 2005 (link)). Supernatants were collected and dried by rotary evaporation. Samples were deacylated by alkaline hydrolysis with methylamine, and lipid extracts were resuspended in 1.0 ml methanol/ methylamine 30%/n-butanol (4:3:1, v/v/v) and incubated 3 h at 55°C in a dry bath (Serunian et al., 1989 (link)). For purification of IPC, the lipid fraction was resuspended in CM/ water (30:60:8, v/v/v; “solvent A”) and applied to DEAE-Sephadex A-25 columns. Columns were washed with five volumes of solvent A and then with five volumes of methanol, and IPCs were eluted with five volumes of 0.05 M sodium acetate in methanol (Toledo et al., 1995 (link)). The acidic fraction was dried, dialyzed exhaustively against distilled water, and partitioned with one volume of n-butanol saturated with water. The butanolic phase was collected and analyzed by HPTLC (Merck) and ESI-MS. IPCs were visualized on HPTLC as blue spots using Dittmer-Lester reagent (Yoneyama et al., 2006 (link)).
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