Silica gel 60 tlc plate

Total lipids were extracted from cells (1 × 10⁶) or liver tissues (50 mg) using the chloroform:methanol (1:2 v/v) extraction method reported by Bligh and Dyer [30 (link)]. Triglycerides and cholesterol were separated using a TLC silica gel 60 plate (Merck, Germany) in a horizontal TLC chamber with an 9 cm separation length with a solution of chloroform:methanol:water (60:30:5 v/v/v). An additional separation from the origin to an 18-cm separation length was achieved with a hexane:diethyl ether:acetic acid (80:20:1.5 v/v/v) solution. TLC plates were visualized by spraying with H₂SO₄, followed by heating at 130 °C for 30 min. Acquired images were processed and analyzed using the Image J program.

DEHU was prepared from alginate using recombinant alginate lyases FlAlyA, FlAlyB, and FlAlex, as described previously^{33 ,44} . An enzyme assay was basically performed in a reaction mixture containing 10 mM sodium phosphate (pH 7.4), 100 mM KCl, 1 mM DTT, 25 mM DEHU, and 0.5 mg mL⁻¹ cell extract or 10–50 µg mL⁻¹ purified enzyme with 5 mM NADH or NAD⁺ at 25 °C. The enzyme reaction was terminated by adding an equivalent amount of chloroform. After vortex-mixing and centrifugation at 10,000g for 5 min, the aqueous phase was subjected to TLC analysis using the TLC silica gel 60 plate (Merck KGaA, Darmstadt, Germany). Products were developed with 1-butanol: acetone: acetic acid: water (35:35:7:23, v:v:v:v) and were visualized by spraying 4.5% (w/v) TBA after the periodic acid oxidation^{19 (link)}.

N-terminal tags (2xHis₆ and Nus) were cleaved off the recombinant ORF1110 protein using HRV3C protease (Novagen) and removed by chromatography on a HisTrapTM FF 1 mL column. The flow-through sample was concentrated to 22.5 μL (7.6 mU/μL) and incubated with 25 μL GM (1 mg/μL) and 2.5 μL acetate buffer (1 M, pH 4.5) at 37°C for 24 h. The sample was then separated by TLC using a TLC Silica gel 60 plate (Millipore) and 1-butanol/ethanol/water (2:1:1, v/v/v) as solvent. For detection the TLC plate was sprayed with 0.2% orcinol and 10% methanol/sulfuric acid and baked at 120°C for 10 min.

The purified ORF1119 protein (3 μg) was incubated with each pNP sugar at 3 mM in 5 μL of 200 mM phosphate buffer (pH 7.5) at 37 °C overnight. The reaction samples were separated by TLC by using a TLC Silica gel 60 plate (Millipore) and 1-butanol/ethanol/water (2:1:1, v/v/v) solvent. The TLC plate was sprayed with 0.2% orcinol and 10% methanol/sulfuric acid, baked at 120 °C, and visualized for spots.

The assay was performed in reference to a previous protocol^{11 (link)}. Briefly, 50 µl of cholesterol solution (20 mM, dissolved in chloroform) and 50 µl cardiolipin (18:1) (20 mM, dissolved in chloroform) were mixed together and dried to form lipid films. Then the films of lipid mixtures were hydrated in 1 ml of 50 mM Tris-HCl, pH 7.4, 160 mM KCl by continuous vortex for 5 min and taken through five cycles of freezing-thawing in liquid nitrogen and a warm water bath at 37 °C. The lipids were extruded 21 times through the 100 nm polycarbonate membranes using an Avanti Miniextruder. 10 µl of above liposomes containing 200 µM cholesterol:cardiolipin (18:1) was mixed with 5 µg of purified ValDLT, ValDLT^H393A, VPA0226, lec/VC_A0218 or VvPlpA in 10 mM HEPES, 150 mM NaCl, pH 7.5, plus 1.4% fat-free albumin in a total reaction volume of 20 µl and incubated at 37 °C for 2 h. The lipids were extracted using the Bligh and Dyer’s method and separated by thin-layer chromatography on TLC Silica gel 60 plates (Sigma) with a mobile phase system containing petroleum ether: ethyl ether: acetic acid in a ratio of 90:10:1. The separated lipid spots were visualized by exposure to iodine vapor.

2-(methylamino)ethanol (MMEA), 2-(ethylamino)ethanol (DMEA), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine sodium salt (LPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (PG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), 1′,3′-bis(1,2-dioleoyl-sn-glycero-3-phospho)-glycerol (CL), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (MMPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl (DMPE) were purchased from Avanti polar lipids. TLC silica gel 60 plates and molybdenum blue spray reagent were purchased from Sigma-Aldrich. All other chemicals used were of analytical grade and commercially available.