Thin layer chromatography membranes

The question as to the potential availability of the requisite amphiphilic precursors in the prebiotic environment has been addressed experimentally by Deamer and coworkers, [143,145] who looked into the uncontaminated Murchison chondrite for the presence of such amphiphilic constituents. Samples of the meteorite were extracted with chloroform-methanol and the extracts were fractionated by thin-layer chromatography, with the finding that some of the fractions afforded components that formed monomolecular films at air-water interfaces, and that were also able to self-assemble into membranous vesicles able to encapsulate polar solutes. These observations dearly demonstrated that amphiphiles plausibly available on the primitive Earth by meteoritic infall have the ability to self-assemble into the membranous vesides of minimum protocells. ... 

We further used a quantitahve procedure to determine the amounts of externalized PS on the surface of apoptohc cells based on its availability to react with fluorescamine, a non-permeating reagent readily interacting with primary amino-groups. Subsequent separahon (by high performance thin-layer chromatography) and quantitahon of fluorescamine-modified PS permits determinahon of absolute amormts of externalized PS on the outer leaflet of plasma membrane. We were also able to determine the amounts of PnA-phospholipids oxidized during apoptosis based on their disappearance in the course of apoptosis. Thus both PS oxidahon and extemalizahon could be assessed. 

Frangopol and Morariu have edited a seminar on procaine and related drugs, methods of analysis, and effects on cell membranes [29]. Items covered include studies on Romanian drugs by mass spectrometry and gas chromatography-mass spectrometry, quantitative and qualitative determination of procaine in biological samples, separation and quantitative thin layer chromatography determination of procaine... 

Figure 9.29 Membrane formation by meteoritic amphiphilic compounds (courtesy of David Deamer). A sample of the Murchison meteorite was extracted with the chloroform-methanol-water solvent described by Deamer and Pashley, 1989. Amphiphilic compounds were isolated chromatographically on thin-layer chromatography plates (fraction 1), and a small aliquot ( 1 p,g) was dried on a glass microscope slide. Alkaline carbonate buffer (15 p,l, 10 mM, pH 9.0) was added to the dried sample, followed by a cover slip, and the interaction of the aqueous phase with the sample was followed by phase-contrast and fluorescence microscopy, (a) The sample-buffer interface was 1 min. The aqueous phase penetrated the viscous sample, causing spherical structures to appear at the interface and fall away into the medium, (b) After 30 min, large numbers of vesicular structures are produced as the buffer further penetrates the sample, (c) The vesicular nature of the structures in (b) is clearly demonstrated by fluorescence microscopy. Original magnification in (a) is x 160 in (b) and (c) x 400.

The enzyme DGAT has not been purified to date, probably because it is a hydrophobic and integral membrane protein. Therefore, DGAT activity was measured using rat liver microsomes as an enzyme source and radiolabeled palmitate as a substrate by the method of Mayorek and Bar-Tana [52] with some modifications [53], The reaction mixture contains microsomal protein, BSA, [14C]palmi-toyl-CoA, MgCl2, diisopropyl fluorophosphate, 1,2-dioleoyl-vw-glyccrol, and a test sample in a total volume of 0.2 ml. After a 15-min incubation at 23°C, lipids are extracted and separated by thin-layer chromatography (TLC). The distribution of radioactivity on TLC is analyzed with a radioscanner to determine the amount of [14C]TG. 

Phospholipids are the major components in membranes therefore they are one of the major contaminants in the initial extraction of LPS. The degree of contaminated phospholipids can be detected by thin layer chromatography (TLC) or mass spectrometry. To remove the contaminated phospholipids, the LPS samples could be washed with chloroform-methanol mixture (1 2, v/v). Phospholipids are soluble in this solvent, but LPS not. 

The methods employed for isolation of the alkaloids depend on the nature of the compounds, and specific conditions have frequently been devised for the selective isolation of particular types of compounds. Usually, fresh or dried plant material is extracted with dilute acid solution or with alcohol, and the extract obtained is further fractionated by extraction into organic solvents with variation of pH. Extraction columns (288), membrane processes (425), and ion-exchange materials (288-290) may be particularly useful for subfractionation or isolation procedures. For further identification and isolation of separate compounds, preparative thin-layer chromatography, (288, 291, 292, 426), liquid chromatography (293, 294), or gas chromatography may be used (202, 296, 297). Because some of the products reviewed in this chapter occur naturally in very small amounts, they have not been isolated in crystalline form. Gas chromatography-mass spectrometry (87, 213, 299), mass fragmentography (192), and mass spectrometry-mass spectrometry (301, 359) have proved to be particularly useful techniques for identification of trace alkaloids in complex mixtures. 

Figure 2. Membranes isolated from Pyrococcus furiosus will phosphorylate Ptdlns to form PtdIns3P. Microsomal membranes were isolated from Daucus carota (wild carrot cells) grown in cell culture and P. furiosus. Lipid kinase activity was assayed as previously described using Ptdlns as a substrate (Bunney et al., 2000). The lipids were extracted and separated by thin layer chromatography as described by Walsh et al. (1991) to separate Ptdlns3P and Ptdlns4P. The migrations of the lipid standards are indicated (Brglez and Boss, unpublished results).

Cell Membrane Labeling. Five million line-10 tumor cells are washed 3 times with 25 ml of PBS, resuspended in 0.4 ml of PBS, and incubated with 10 /ag of lactoperoxidase, 3 mU of glucose oxidase, 2.5 yM glucose (0.23 /xg), and 100 /xCi of Na I in a total volume of 0.5 ml for 15 min at ambient temperature. The cells are washed 5 times with 10 ml of PBS, and the lipid and nonlipid fractions of the cells are obtained through a Folch extraction. The cellular lipids are separated by thin layer chromatography. ... 

The fatty acid composition of lipids is usually analyzed by gas chromatography following transesterification into methyl esters. Unmodified lipids can be analyzed by HPLC or by soft chemical ionization mass spectrometry. In the course of sample preparation it is often necessary to separate the various membrane fractions (plasma membrane, thylakoid, microsomal, mitochondrial, etc.) by sophisticated gradient centrifugations, as well as the individual lipid classes within a membrane fraction, usually by thin-layer chromatography (TLC). 

As emphasized, dietary fatty acids produced drastic modification of the molecular species of brain ethanolamine phospholipids. Hargreaves and Clandinin reported similar findings in the rat (Connor et al., 1997). Using argentation thin-layer chromatography (TLC), which is unable to resolve individual molecular species, they fed fish-oil or linseed-oil diets to rats, which resulted in an increased microsomal and synaptic membrane content of phosphatidylethanolamine species containing six double bonds, and a decrease in species containing five double bonds, compared with animals fed soy or safflower oil. 

Test for phospholipase A activity. Acetone powders of membrane fractions were incubated with [3H]-lecithin at 37°C. The incubation mixture was extracted with methanol-chloroform, the extracted solution was washed and concentrated and the labelled phospholipids were separated and identified by thin layer chromatography in the presence of authentic reference compounds. The spots were scraped off and their radioactivity was measured. It was found that the membrane powders had formed some [3H]-lysolecithin. 

The formation of thin layers and membranes, for analytical applications or as sensors, has also been studied. Here, either the MIP is grown directly on a surface (for instance an electrode) [115], or particles of MIP are held together by a binder to generate a material similar to those used for thin layer chromatography [ 116]. The first method avoids the grinding step and thus reduces the risk of deforming the sites. 

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