The lipids obtained from 2.88 × 105 cells, as described above, were suspended in 600 μl 0.1 M KOH in chloroform-methanol (2:1 [vol/vol]) and incubated for 2 h at 37°C. After incubation, the lipid solution was sequentially mixed with 21 μl 4 M formic acid, 200 μl chloroform, and 400 μl deionized water. Then, the phases were separated by centrifugation at 770 × g for 5 min at ambient temperature, and the organic phase was recovered, dried, and dissolved in 50 μl chloroform-methanol (1:1 [vol/vol]) (47 (link)). The obtained lipids were resolved by TLC on Silica Gel 60 high-performance TLC plates (Merck, Darmstadt, Germany) with chloroform-methanol-15 N NH3 (60:35:8 [vol/vol/vol]). Each spot on the TLC plates was analyzed as described above.
High performance tlc silica gel 60 plates
Manufactured by Merck Group
Sourced in Germany, United Kingdom
High-performance TLC silica gel 60 plates are a type of laboratory equipment used for thin-layer chromatography (TLC) analysis. These plates are made of silica gel 60, a commonly used stationary phase in TLC. The high-performance characteristics of these plates provide efficient separation and resolution of analytes during TLC experiments.
Automatically generated - may contain errors
Lab products found in correlation
Autoradiography film, by Kodak (2 mentions)
1 14c acetate, by Cytiva (2 mentions)
Phosphorimager screen, by Cytiva (2 mentions)
Imagequant software, by Cytiva (2 mentions)
Imagequant tl, by Cytiva (2 mentions)
Storm 820 phosphorimager, by Cytiva (2 mentions)
Fuji imaging analyzer, by Fujifilm (1 mentions)
3h sphingosine, by PerkinElmer (1 mentions)
1 14c acetate, by PerkinElmer (1 mentions)
Bas 2500, by GE Healthcare (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
35s sulfate, by Cytiva (1 mentions)
8 protocols using high performance tlc silica gel 60 plates
1
Lipid Extraction and Separation by TLC
2
Lipid Synthesis in E. invadens Trophozoites
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E. invadens trophozoites suspended in proliferation medium (1.5 × 105/ml) or encystation medium (6 × 105 cells/ml) were seeded in 96-well culture plates (240 μl per well). After adding U-14C-labeled l -serine (173.6 mCi/mmol) (Moravek, Brea, CA, USA) to each well (final radioactivity, 3 μCi/ml), the plates were sealed and incubated at 26°C for the period indicated as described above. For each time indicated, cell cultures from four wells of a 96-well plate were collected in a single 6-ml glass tube, and cells were pelleted by centrifugation at 1,500 × g for 5 min at 4°C. The cell pellet in each tube was washed twice with PBS. Then lipids were extracted by successive addition of 3.8 ml chloroform-methanol-0.15 N HCl (5:10:4 [vol/vol/vol]), 1 ml chloroform, and 1 ml 1% KCl (wt/vol deionized water) with thorough mixing at each addition. Phases were separated by centrifugation at 770 × g for 5 min at ambient temperature, and the organic phase was recovered and dried. The lipids extracted from 2.88 × 105 cells were resolved by thin-layer chromatography (TLC) on Silica Gel 60 high-performance TLC plates (Merck, Darmstadt, Germany) with chloroform-methanol-15 N NH3 (60:35:8 [vol/vol/vol]). Each spot on the TLC plates was quantified using a Fuji imaging analyzer and Multi Gauge 2.2 software (FLA-7000; Fujifilm, Tokyo, Japan).
3
Sphingosine Labeling Assay Protocol
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Sphingosine labeling assay was performed as previously described (Nakahara et al., 2012 (link)). Cells were incubated for 12 h at 37°C with DMEM containing charcoal-treated 10% FCS and for another 12 h with DMEM. Cells were then labeled with 0.6 μCi of [3H]sphingosine (PerkinElmer Life Sciences, Waltham, MA) for 4 h at 37°C. Lipids were extracted, separated by normal-phase TLC on Silica Gel 60 high-performance TLC plates (Merck Millipore, Billerica, MA) with 1-butanol/acetic acid/water (3:1:1 [vol/vol]), and visualized by fluorography.
4
Bacterial Lipid Composition Analysis
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The lipid compositions of bacterial strains were determined following labeling with [1-14C]acetate (Amersham Biosciences). Cultures (1 ml) of Burkholderia s.l. strains were inoculated from precultures grown in the same medium. After addition of 1 μCi of [14C]acetate (60 mCi mmol-1) to each culture, the cultures were incubated overnight. Cells were harvested by centrifugation, washed with 500 μl water once, resuspended in 100 μl water, and then lipids were extracted according to Bligh and Dyer (Bligh and Dyer, 1959 (link)). Aliquots of the lipid extracts were spotted on high performance TLC silica gel 60 plates (Merck, Poole, UK) and separated in two dimensions using chloroform/methanol/water (16:4:1, v/v/v) as a mobile phase for the first dimension and chloroform/methanol/acetic acid (15:3:2, v/v/v) as a mobile phase for the second dimension (Tahara and Fujiyoshi, 1994 (link)). To visualize membrane lipids, developed two-dimensional TLC plates were exposed to autoradiography film (Kodak) or to a PhosphorImager screen (Amersham Biosciences). The individual lipids were quantified using ImageQuant software (Amersham Biosciences) (Vences-Guzmán et al., 2011 (link)).
5
Lipid Composition Analysis of E. coli Strains
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The lipid composition of the different E. coli strains was determined by triplicate following labeling with [1-14C]acetate (Perkin Elmer). LB cultures (1.5 mL) were inoculated to an initial optical density at 600 nm (OD600) of 0.1 from precultures grown in the same medium. After the addition of 1 μCi/mL [1-14C]acetate to each culture, they were incubated for 24, 48 or 72 h. The cells were harvested by centrifugation and resuspended in 100 μL of water. The lipids were extracted according to the method of Bligh and Dyer [51 (link)]. The chloroform phase was used for lipid analysis by one-dimensional thin-layer chromatography (TLC) using high-performance TLC silica gel 60 plates (Merck) and ethyl acetate-hexane-acetic acid (60:40:5 [vol/vol/vol]) as the mobile phase. Two-dimensional TLC was performed as described previously [52 (link)]. Radioactivity was detected using a Storm 820 PhosphorImager (Amersham Biosciences). Image analysis and signal quantification were carried out using ImageQuant TL (Amersham Biosciences).
6
Radioactive Lipid Labeling Protocol
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[32P]S1P was prepared using [γ-32P]ATP (6000 Ci/mmol; PerkinElmer Life Sciences), Sph (Avanti Polar Lipids), and total cell lysates prepared from human embryonic kidney 293T cells overexpressing the Sph kinase SPHK2, as described previously (50 (link)), and dissolved in ethanol. [32P]Pi and [32P]S1P labeling assays were performed as follows. One hour before labeling, the medium containing FBS and antibiotics was replaced with medium devoid of them. Cells were labeled with 0.2 μCi [32P]Pi (phosphorus-32; 8500–9120 Ci/mmol; PerkinElmer Life Sciences) or 0.2 μCi [32P]S1P in the presence or the absence of fatty acid-free BSA for 1 or 3 h. After removing the medium, cells were washed twice with PBS containing 1% BSA. PBS was added to cells, and cells were detached from the dishes using a scraper, centrifuged, and suspended in PBS. Lipids were extracted as described previously, dried, suspended in chloroform/methanol (1:2, v/v), and separated by normal-phase TLC using high-performance TLC silica gel 60 plates (Merck) and 1-butanol/acetic acid/water (3:1:1, v/v) as a solvent system. 32P-labeled lipids were detected and quantified by an imaging analyzer BAS2500 (GE Healthcare Life Sciences).
7
Lipid Profiling of S. meliloti and E. coli
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The lipid composition of the different S. meliloti and E. coli strains was determined following labelling with [1-14C]-acetate as previously described [3 (link)]. Lipids from cell pellets were extracted according to the method of Bligh and Dyer [29 (link)] and lipids from spent media supernatants were extracted with equal volumes of acidified ethyl acetate (0.1 mL glacial acetic acid per litre of ethyl acetate). Lipids obtained were analysed by one-dimensional thin-layer chromatography (TLC) using high-performance TLC silica gel 60 plates (Merck) and mobile-phase ethyl acetate-hexane-acetic acid (60:40:5 (v/v/v)). Radioactivity was detected using a Storm 820 PhosphorImager (Amersham Biosciences). Image analysis and signal quantification were carried out using ImageQuant TL (Amersham Biosciences). E. coli BL21 (DE3)-derived strains were grown in M9 MM, and protein expression was induced by the addition of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) during the mid-exponential phase of bacterial growth (OD620nm = 0.4). The cultures were collected 21 h after induction with IPTG. For labelling experiments, S. meliloti strains were grown on Robertsen MM. Cultures were labelled at OD620nm = 0.1 and collected after 66 h of growth. For each strain, labelling experiments were repeated 3 times and representative TLCs are shown.
8
Bacterial Lipid Composition Analysis
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