Folch wash

Quantification of hexose units in glycolipids can be carried out by making the furfural derivatives with strong acid and reacting them with anthrone to yield coloured compounds (Yamamoto and Rouser, 1970). a-Naphthol will also give coloured derivatives (Yamamoto and Rouser, 1970). Ganglioside-calcium complexes are insoluble and this method can be used to ensure that they are extracted into the lower phase of Folch washes (Carter and Kanfer, 1975). The AT-acetylneuraminic acid content of... 

Derivatives are often used in lipid chemistry to prepare fatty acid methyl esters needed to determine the fatty acid composition of lipids. Fried et al. (1992) published a technique on the transesterification of 500-mg samples of snail tissue. Because the technique is applicable to other biological tissues and fluids, it is presented herein. Lipids were extracted from snail bodies with 10 ml of chloroform-methanol (2 1) the extracts were filtered through a plug of glass wool contained in a Pasteur pipet, and nonlipid contaminants were removed by extraction with 8 ml of Folch wash (0.88% aqueous KCl). The lipid-containing lower phase was separated and evaporated just to dryness under a stream of nitrogen at room temperature. The total lipid sample was dissolved in about 30 ml of methanol, and 0.5-1.0 ml of concentrated sulfuric acid was added. The mixture was refluxed for 1 h the formed fatty acid methyl esters were extracted with 30-40 ml of petroleum ether and the extract dried over anhydrous sodium sulfate. The fatty acid methyl esters were concentrated on a Rotoevaporator at 40°C and the volume reduced to 1 ml. The fatty acid methyl esters can be separated by TLC on silica gel impregnated with silver nitrate (Christie, 1982). 

Extraction of Plant Sampks With plant tissues, it is necessary to extract first with isopropanol in order to deactivate the enzymes, and a procedure devised by Nichols is usually recommended [44]. The plant tissues are macerated with 100 parts by weight of isopropanol. The mixture is filtered, the solid is extracted again in a similar manner and finally is shaken overnight with 199 parts of chloroform-isopropanol (1 1, v/v). The combined filtrates are taken up in chloroform-methanol (2 1, v/v) and given a Folch wash as described earlier. The purified lipids are recovered from the lower layer as described in Folch extraction. 

The search for lipids containing amino acids in a lipidic bacterial extract makes it necessary to exclude the presence of free amino acids or peptides that can frequently exist as contaminants in lipid extracts. Water-soluble contaminants can be removed by washing a chloroform phase with methanol-water ( Folch wash ) (5) or by passage of a chloroform solution of the crude extract through a column of Sephadex G-25 4,5). 

Prior to phospholipid analysis, it is imperative to extract the lipids from their matrix and free them of any nonlipid contaminants. Phospholipids are generally contained within the lipid fraction, which may be recovered by the traditional Bligh and Dyer or Folch extraction procedure (9,22). In any phospholipid extraction method it is recommended to include a rather polar solvent in addition to a solvent with high solubility for lipids. The former is needed to break down lipid-protein complexes that prevent the extraction of the lipids in the organic phase. Traditionally, mixtures of chloroform and methanol (especially 2 1, v/v) have been recommended. These are washed with water or aqueous saline to remove nonlipid contaminants. Comparing the recovery of phospholipids, Shaikh found that the neutral phospholipids PC, PE, SPH as well as DPG were nearly quantitatively extracted by all solvent systems studied (Table 1), although Bligh and Dyer, in which the lower phase was removed only once, was somewhat worse (23). 

However, Shaikh demonstrated that the aforementioned traditional methods are inappropriate to recover completely lysophospholipids as well as acidic phospholipids classical Folch gave 85-90% recovery of LPC and LPE, whereas Bligh and Dyer yielded only 75-80% recovery. Extraction with a mixture of chloroform and methanol, on the other hand, provided nearly complete recovery of acidic and lysophospholipids, but up to 15% losses were observed during subsequent washing, according to Folch. These losses could be circumvented by purification of the crude extract on Sephadex G-25, but this column chromatographic procedure is quite time-consuming. 

In order to prevent the formation of a stable emulsion at any stage of the extraction procedure, the water content of the hydrated WPC has to be controlled so as not to obtain a biphasic solvent system during extraction with mixtures of chloroform and methanol. Besides, nonlipid contaminants are removed from the extract by gel filtration on nonlipophilic Sephadex G-25 instead of traditional aqueous washing total lipids were eluted with a 19/1 (v/v) mixture of chlo-roform/methanol, saturated with water, whereas a 1/1 (v/v) mixture of water and methanol eluted nonlipid contaminants. The method yields a similar total lipid content to the Folch method, but it is about four times faster (24). 

The neutral lipid fraction from the DEAE-Sephadex A-50 column was combined with the lower phase obtained after Folch partition of the total lipid extract and the combined lipids dried. To the same flask, 10Q ml of 0.6 M NaOH in methanol was added. The mixture was incubated at 37°C for 5 hours. Five volumes of acetone were then added and stored overnight at 4°C. The precipitate was collected by centrifugation at 4°C and dissolved in C M (4 1, v/v). After application to the column (2.0 x 25 cm), the column was washed with chloroform. Neutral glycolipids were then eluted with tetrahydrofuran H2O (10 1). Fractions containing neutral glycosphingolipids were pooled and their glycolipid content examined by thin-layer chromatography. 

Currently, there is no doubt that the most widely used method for extraction of tissue lipids is that of Bligh and Dyer (1959). Basically, this is a modification of the Folch method and employs a careful calculation of the amount of sample (tissue) water such that the overall mixture will have a final composition of chloroform-methanol-water of 1 2 0.8 (v/v). Thus, a singlephase extract can be obtained and extraction completed very rapidly, even within minutes. Recovery of the lipid in a chloroform-rich phase can be achieved by addition of equal volumes of chloroform (under certain conditions) and water to produce a two-phase system. The lower (CHC13) phase is subsequently washed with a methanol-water (1 0.9, v/v) mixture to allow removal of a substantial amount of the nonlipid contaminant with little or no problems with interfacial fluff formation or emulsions. However, even though this is a highly efficient method, it is still advisable that one take steps... 

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 unusual solubility behavior of, for example, the Tay-Sachs brain tissues is due to qualitative and quantitative differences in the composition of the appropriate lipid.211 The isolation and purification of these anomalous gangliosides were carried out similarly to those for the gangliosides of normal tissues, for example, by the method of Folch and coworkers.182 In this method, the brain tissues are extracted with chloroform-methanol-water, the extract is washed with potassium chloride solution, and the extracted material is separated on a column of silicic acid by elution with an increasing concentration of methanol in chloroform. 

Metabolism of CLN in Intestinal Epithelial Cells. Caco-2 cells (5 X 10 cells/dish) were precultivated in a 55-cm tissue culture dish for 24 h. Then, c9,tll,tl3-CLN was added to Ae culture medium as 10% AmeAyl sulfoxide (DMSO) solution. The final concentration of DMSO never exceeded 0.1% (v/v). After incubation, cells were trypsinized and washed three times with phosphate buffered saline. The TL of cells was extracted by Ae Folch s procedure (23). 

Frozen myocardium was ground to powder and H-myocardial hpids were extracted with chloroform imethanol (Folch). After washing, the lipids were re-solubilised in chloroform and separated by TLC using a hexane-diethylether-acetic acid system. Lipid classes were coimted for H-radioactivity. 

Lipid extracts were prepared and washed according to Folch et al, (1957), then were dried under N2 and applied as oblique bands on preparative two-dimensional TLC plates (Rodriguez de Turco Ba-zSn, 1977). The lipids were scraped off and eluted according to Arvidson (1968). Neutral lipids were rechromatographed as described elsewhere (Aveldaho Bazan, 1974). 

Numerous extraction procedures are available for lipids, and this subject has been reviewed by Phillips and Privett (1979), Christie (1984), and Fried (1993). Of all extraction procedures, that of Folch et al. (1957) is used most frequently. This procedure involves extracting fresh animal or plant material with chloroform-methanol (2 1), usually in a volume ratio of 20 parts of the solvent to 1 part tissue or fluid. There are many variations of the Folch et al. (1957) procedure, and for descriptions of these variations, see Christie (1982, 1984). Experiment 4 involves the use of the Folch et al. (1957) procedure on 100 mg of animal tissue and 100 pi of serum. The Folch et al. (1957) procedure extracts nonlipid material along with lipids. Nonlipids are usually removed by an aqueous wash with either water or a dilute salt solution. Phillips and Privett (1979) devised an extraction procedure for brain tissue in which nonlipid material is first removed with dilute acetic acid. The brain tissue is then treated with chloroform-methanol essentially as described in Folch et al. (1957) to get a relatively pure lipid fraction. Because so many different lipid extraction procedures have been used prior to TLC analysis, different results for a given separation may reflect technique differences rather than acmal differences in lipid constituents of a sample. 

Lipid extracts obtained from biological samples, as aforementioned, tend to contain significant amounts of nonlipid contaminants, such as sugars, amino acids, urea, and salts. These must be removed before the lipids are analyzed. A common and classical approach is to use a simple washing procedure devised by Folch, Lees and Sloane Stanley [17], in which a chloroform-methanol (2 1, v/v) extract is shaken and equilibrated with one-fourth its volume of saline solution (i.e., 0.88% potassium chloride in water). The mixture partitions into two layers, of which the lower phase is comprised of chloroform-methanol-water in a proportion of 86 14 1 (by volume) and contains virtually all of the lipids, while the upper phase consists of the same solvents in the proportion of 3 48 47 (by volume), respectively, and contains much of the nonlipid contaminants. It is important that the proportion of chloroform, methanol, and water in the combined phases should be as close as possible to 8 4 3 (by volume), otherwise selective losses of lipids may occur. If a second wash of the lower phase is needed to remove any remaining contaminants, a mixture of similar composition to that of the upper phase should be used, i.e., methanol-saline solution (1 1, v/v). 

Folch and Van Slyke (33) removed the nitrogenous nonlipid materials from plasma by treating the sample with colloidal iron which precipitated the proteins and lipids together. Water-soluble contaminants were then washed out from the precipitate with watet and an aqueous solution of magnesium sulfate, followed by extraction of the lipids from the moist residue with a mixture of alcohol and ether. For complete extraction of the lipids, the presence of water was necessary in a ratio of 1 volume of water to 6 volumes of the alcohol-ether mixture. 

Samples of leaves of corresponding age, harvested from replicate sets of stressed and control plants, were weighed, extracted with boiling isopropanol, followed by chloroform methanol (2 1 then 1 2, v/v) and washed by the procedure of Folch et al. (1957) to separate the lipid fraction. Thin layer chromatography and estimations of lipids were carried out as described previously (L5sel and Lewis, 1974). In studies of incorporation of C-labelled photosynthate into lipids, 0.5 g samples of leaves were laid on damp filter paper in a transparent plastic box, allowed to photosynthesize in for 30 minutes and for... 

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