Folch extraction

1 Folch Extraction Many modifications of the basic extraction procedure 

Obviously, a reduction in sample and solvent for this procedure of lipid extraction of biological samples for lipidomics analysis is necessary. The key in the procedure is to use methanol to homogenize the tissue sample and use chloroform-methanol (2 1, v/v) to dissolve the lipids. As used only for small sample materials in the case of lipid extraction for lipidomics, the water content present in the original samples can essentially be neglected in the phase separation procedure. 

3 Greys Method for Phosphatidyl inositol Phosphate Extraction 

Grow tissue cultures in appropriate media in 10cm cell culture dishes until 60-80% confluent for adherent cells or suspension cells. 

For adherent cells, keep the culture dish on a bed of ice during cell harvest and aspirate media from dish. For suspension cells, spin the cells at 1700 rpm for 5 min. 

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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). 

As far as the acidic phospholipids are concerned, the recovery of PI, PS, PA, and PG was limited to only 30-50% by Bligh and Dyer extraction, whereas Folch extraction yielded about 85% recovery of PI and PS but only 20% and 35% recovery of PA and PG, respectively (Table 1). The poor recovery of acidic PL is thought to be due to adsorption of PL to proteins in a neutral... 

Plasma, cells, tissues Modified Folch extraction, sonication, homogenization UPHLC (C18) MS ESI hybrid quadrupole time of flight 18 (39)... 

Egg, soya, and porcine Folch extraction, filtration 2D HPLC (ID HILIC/silica, 2D C18) MS ESI ion trap 260 (42)... 

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 lipid fraction of the supernatants is obtained through a Folch extraction, and the lipid classes are separated by analytical thin layer chromatography. The amount of activity associated with each separated lipid moiety is then quantified. Treatment of line-10 cells with antibody plus... 

The preparation of a lipid sample includes extraction of the lipid material from the examined object (seeds, tissues, food, etc.), choosing among the several widely accepted procedures. The extraction with chloroform-methanol (2 1) (the Folch extraction) is the most popular. A solution of known concentration in hexane or di-chloroethane is prepared and a suitable aliquot is applied on the plate as a small spot or, better, as a narrow band. Two, three, or more solvents, mixed in different proportions, give the mobile phase. Development is performed in common tanks (Desaga type, for example), in the ascending mode. For fine separations, cylindrical or sandwich-type tanks provide better results. 

Fatty acids can be extracted either as FFAs or as BFAs. FFAs are obtained by the hydrolysis of BFAs. Liquid-liquid extraction has been successfully applied in the isolation of several classes of lipids, or of single lipids from complex mixtures. Popular extraction methods for lipids are the Folch extraction technique, or the Bligh and Dyer method. Solid-phase extraction (SPE), and solid-phase microextraction (SPME), are also available as simple and economical time- and solvent-efficient sample preparation methods. Prefractionation can be performed using SPE silica or aminopropyl-silica columns. 

Prepare lipid mixtures to a total lipid concentration of 0.1 mg/ml in a 19 1 mixture of chloroform methanol. Methanol is necessary to maintain the solubility of some lipids, including PtdIns(4,5)P2. The lipid mixture can be tailored to the protein of interest, with 10% cholesterol, 40% PtdEth, 40% PtdSer, and 10% PtdIns(4,5)P2 used for the analysis of AP180 and epsin mediated clathrin nucleation. An alternative is to use a brain lipid extract such as Folch extract. Lipid mixtures should be prepared fresh. 

Extraction in 20 vol. of ethanol as described above is likely to give the most complete recovery of all types of bile acids. Alkaline conditions favor extraction. Folch extraction has been used to get a simultaneous purification, and the conjugated bile acids are then found in the upper phase (20). A different type of extraction is that described by Hofmann, who used a liquid anion exchanger, tetraheptylammonium chloride (21). This was added to bile and the salts formed with bile acids could be quantitatively extracted with ethyl acetate. The use of this solvent makes the evaporation of bile extracts simple. The amine can be removed with a cation exchanger, a procedure first described by Anderson (22). 

Intestinal contents can be extracted with ethanol or chloroform/ methanol (1, 20). Shioda et al. (20) used a Folch extraction for duodenal bile and recovered the conjugated bile acids in the top water phase. Free bile acids are often present in intestinal contents (usually distally) and may be partly lost in this procedure. 

In the Folch extraction procedure ground or homogenized tissue is shaken with a 2 1 mixture of chloroform and methanol and the organic extract is subsequently partitioned with aqueous potassium chloride solution. It is important that the ratio of chloroform, medianol and water in the final mixture be 8 4 3. The extracted lipids are in the (lower) chloroform layer. 

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. 

This method is modified for lipid extraction for lipidomic analysis. For example, Welti et al. omitted the filtering step and directly extracted the lipids from plant tissues by Folch extraction method [45]. 

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