Cells were grown at 30°C to mid–log phase in 250 ml YPDA medium and collected. Total lipids were extracted by the Bligh and Dyer method (Bligh and Dyer, 1959 (link)). Phospholipid amounts were determined by phosphorus assay (Rouser et al., 1970 (link)). For the phospholipid analysis, samples containing 200 nmol phosphates were subjected to TLC plates (Merck, Darmstadt, Germany), and phospholipids were detected as described previously (Mioka et al., 2018 (link)). To detect free and esterified ergosterol, lipid extracts containing 20 nmol phosphates were subjected to high-performance TLC (Merck) separation with hexane/diethyl ether/formic acid (40:10:2, vol:vol:vol). Ergosterols were stained with a mixture of ferric chloride/sulfuric acid/acetic acid by heating (Lowry, 1968 (link)), and the spots were scanned by an image analyzer. The ergosterol content was determined by TLC-densitometric analysis using ImageJ.
High performance tlc
Manufactured by Merck Group
High-performance TLC (Thin-Layer Chromatography) is a laboratory instrument used for the separation and analysis of chemical compounds. It provides a reliable and efficient method for the separation and identification of complex mixtures. The core function of this equipment is to facilitate the separation and analysis of various substances through the process of thin-layer chromatography.
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Lab products found in correlation
2 protocols using high performance tlc
1
Quantification of Yeast Lipid Profiles
2
Lipid Extraction and Cholesterol Analysis
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Cells were collected in an ice-cold buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 1 mM EGTA, 2 mM DTT, 200 mM sucrose) containing protease inhibitors (25955-11, nacalai tesque), and phosphatase inhibitors (8 mM NaF, 12 mM β-glycerophosphate, 1 mM Na3VO4, 1.2 mM Na2MoO4, 5 µM cantharidin, and 2 mM imidazole), homogenized with 2 passages through a 27-gauge needle after 6 passages through a 23-gauge needle, and centrifuged at 3000 × g for 5 min at 4 °C. The post-nuclear supernatant was overlaid on 10 µL of 2 M sucrose and centrifuged at 100,000 × g for 1 h at 4 °C. Total lipids were extracted by the Bligh and Dyer method68 (link). To detect cholesterol, lipid extracts were subjected to high-performance TLC (Merck) separation with hexane/diethyl ether/ acetic acid (80:20:1, vol:vol:vol). Cholesterols were stained with a mixture of ferric chloride/sulfuric acid/acetic acid by heating69 (link).
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