Chloroform/methanol precipitation

Protein is precipitated to get rid of ions or agents that interfere with the protein determination or gel electrophoresis, and/or to concentrate the protein. The method of choice for samples less than 500 J,1 is the chloroform/methanol precipitation. However, the native conformation of the proteins gets lost. 

Chloroform/methanol precipitation Wessel and Flugge (1984) dilute watery protein solutions (volume of 10 to 150 pi) in Eppendorf tubes with methanol and precipitate the proteins with chloroform. Addition of water separates the water/methanol/chloroform solution into two phases. The precipitated proteins collect in the interphase. Test volumes of 0.2 to 2 ml can also be processed with Corex glass tubes. 

Remove the excess of probe by chloroform/methanol precipitation. 

After stirring for 1 hr more, the reaction mixture is diluted with 500 ml of cold water the yellow precipitate which forms is filtered, washed and dried, to give 4.1 g (83%) of crude 16-diazoestrone methyl ether (94) mp 133-136°. Chromatography of the crude product over neutral alumina in chloroform, followed by recrystallization from chloroform-methanol, gives pure (94) mp 145-146°, in 64% over-all yield. ... 

Isolation of Sesquiterpene Lactones. The ether extract was evaporated and dissolved in 952 ethanol. Then an equal volume of 42 aqueous lead acetate was added. After 1 hour the mixture was filtered to remove precipitated chlorophyll and phenolic products and the ethanol removed under vacuum. The aqueous layer was extracted with chloroform giving a dark colored oil from which the sesquiterpenes were isolated by a combination of chromatographic procedures, i.e., LH-20 gel permeation, silica gel using both packed columns and thin layer plates. A variety of solvents were also used to purify the individual sesquiterpene lactones, e.g., benzene-acetone (1 1), ethyl acetate, chloroform-methanol (9 1). On thin layer chromatographic plates, spots were visualized by spraying with 22 aqueous KMn04 solution. 

To a solution of triphenyl phosphite (6.2 g, 0.02 mol) and thiomethoxy-acetaldehyde (2.25 g, 0.025 mol) in glacial acetic acid (18 ml), powdered N-phenylthiourea was added in a single portion. The reaction mixture was stirred at room temperature for 30 min and then for 30 min at 80°C. After the mixture was cooled to room temperature, water (5 ml) was added and the solution was maintained at room temperature for 10 h. The precipitate was removed by suction filtration, washed with 1 1 acetic acidtwater (2 x 10 ml), dried over potassium hydroxide in an evacuated dessicator, and recrystallized from chloroform/ methanol. In this manner there was isolated pure 0,0-diphe-nyl 2-methylthio-l-(iV-phenylthioureido)ethylphosphonate (8.61 g, 94%) of mp 136 to 138°C, which exhibited spectra and analytical data in accord with the proposed structure. 

It is commonly known that lipids, carbohydrates, and glycolipids are present in the Golgi apparatus (27). The determination of the components that react with the ZIO mixture was carried out by removing each component from tissues before incubation in the ZIO mixture. After lipid extraction by acetone (14), chloroform-methanol (15), or propylene oxide (27), no osmium-zinc precipitates could be detected in structures that normally reacted with ZIO. Blumcke et al. (15) summarized the nature of the lipids that react with the ZIO mixture as follows lipids and lipoproteins of cell membranes, neutral fat droplets (41), and lipid globules of type II pneumocytes and alveolar macrophages were, however, not as electron dense as the normally reactive lamellae containing highly unsaturated fatty acids. 

Lipids can be extracted from biological samples using a variety of organic solvents. A chloroform-methanol solvent is suitable for all lipids but it is possible to extract different classes of lipid selectively on the basis of their solubility in different organic solvents. This may be achieved by the addition of a solvent that will effect either the precipitation or the extraction of the lipids of interest. An example of the former is the precipitation of high concentrations of phospholipids with cold, dry acetone, and of the latter, the extraction of fatty acids into ether or heptane at an acid pH. However, like all solvent extraction procedures these are not entirely specific. 

To a stirred solution of 0.02 mole of an orange-red solution of potassium diphenylmethide in 150 ml of liquid ammonia and 50 ml of dry ethyl ether, prepared from 0.02 mole of KNH2 and 3.36 gm (0.02 mole) of diphenyl-methane, is added 2.5 gm (0.01 mole) of l,l-dichloro-2,2-diphenylethene. The reaction mixture darkens and then a precipitate forms. The reaction mixture is stirred until all the ammonia evaporates to leave a solid. The solid is recrystallized from chloroform-methanol to afford 3.1 gm (90%) of tetraphenylallene, m.p. 164°C. 

A reaction kettle equipped with mechanical stirrer was placed in a glove box filled with nitrogen and heated to 120°C for 1 hour to remove moisture. The reactor was then charged with glycolide (200 g), y-caprolactone (200 g), 0.27 ml dodecanol, 700 ml of xylene, and 1.12 ml of stannous octoate and where glycolide component was added over four portions in 2-hour intervals. The mixture was then stirred at 120°C for 90 hours and the product isolated after dissolving in chloroform and precipitated in methanol. 2... 

The bottom chloroform layer was filtered and allowed to concentrate to a final volume of 5 mL. Pure PHA was obtained by nonsolvent precipitation (chloroform methanol in a ratio of 1 9). Finally, the white precipitate was dried and weighted. 

To a solution of 5.5 g of 3-chlorsulfonyl-4-chlorobenzoylchloride in 20 ml chlorobenzole 3.3 g of l-amino-cis-2,6-dimethylpiperidin-hydrochloride slowly was added at room temperature. Reaction mixture was heated to 100°-105°C during 8 h, then it was cooled to room temperature. The precipitated crystals of N-[cis-2,6-dimethyl-piperidyl-(l)]-3-chlorsulfonyl-4-chlorobenzoylamide were filtered and dried at 100°C under vacuo, then was treated by ammonium at room temperature. The obtained product was chromatographed (aluminum oxide eluent chloroform-methanol 9 1 and methanol-ether) to give N-[cis-2,6-dimethylpiperidyl-(l)]-3-sulfamyl-4-chlorbenzoylamide, melting point 235°-237°C. 

In order to make the separation of unreacted cellulose from the apparent graft easier it was acetylated under nondegradative conditions in acetic anhydride-pyridine mixtures. The apparent graft copolymer was first soaked in methanol, methanol was replaced by water and then acetylated with a 1 2 acetic anhydride-pyridine mixture for 36 hr at 100 °C. After the reaction, the product was precipitated with n-hexane. The precipitate was redissolved in chloroform-methanol and reprecipitated with methanol. The reprecipitation was repeated and finally a purified acetylated product was obtained. When pure cellulose was used for the acetylation it was confirmed that by this procedure cellulose is converted almost totally (98.5 to 99,6%) to triacetate. 

After the seromucoids were precipitated with phosphotungstate-HC1, they were exhaustively extracted with hot alcohol-ether (3 to 1 by volume), followed by two hot extractions with chloroform-methanol (1 to 1). The combined solvent extracts were evaporated on the steam bath under nitrogen and the lipides taken up with petroleum ether. The petroleum ether extracts were washed several times with water containing acetate and 0-hydroxybutyrate and acetoacetate (10). 

The lower, chloroform-rich phase is separated carefully from the protein-containing interface, and then it is washed twice with methanol-water (10 9, v/v) and the washes are discarded. The chloroform layer contains the phosphatidic acid (as a sodium salt) and can be isolated by acetone precipitation. The yields can be of the order of 90-95%. One alternative route to identification of the chloroform-soluble material is to analyze it for total phosphorus and total fatty acid ester (see procedures described earlier). In the case of diacylphosphatidylcholine as the substrate, the fatty acid ester/P molar ratio should be 2.0. Another approach is to subject the chloroform-soluble fraction to preparative thin-layer chromatography on silica gel H (calcium ion free) in a two-dimensional system with a solvent system of chloroform-methanol-28% ammonium hydroxide (65 35 6, v/v) in the first direction and a solvent system of chloroform-acetone-methanol-glacial acetic acid-water (4.5 2 1 1.3 0.5, v/v) in the second direction. The phosphatidic acid will not migrate far in the basic solvent Rf 0.10) and will show an Rf value one-half of that of any remaining starting substrate (fyO.40) in the second solvent. Of course with a simple substrate system, one can use the basic solvent in one dimension only... 

Extraction. To 1 ml of tissue homogenate is added 3.75 ml of chloro-form/methanol solution so that the ratio of chloroform/methanol/ water in the mixture is 5 10 4 (v/v/v). After thorough mixing, the mixture is converted to a biphasic system by addition of 1.25 ml of chloroform and a similar volume of potassium chloride reagent so that the ratio of components is 10 10 9 (v/v/v). After centrifugation at 2000 x g for 10 min, the upper phase is carefully aspirated so that the precipitate which forms at the interphase is undisturbed and a further 4.58 ml of the water chloroform/methanol mixture is added. After mixing, the phases are separated by centrifugation and the lower phase collected quantitatively into a clean stoppered test-tube and evaporated under N2 at 20°C. 

The incubation mixture contained ornithine, pyridoxyl phosphate, and the enzyme. After incubation for 1 hour, the reaction was terminated with perchloric acid. The precipitate was removed by centrifugation, the supernatant extracted with chloroform-methanol (2 1), and the aqueous layer applied to a CellexP column. The putrescine was eluted, reacted with fluorescamine, and quantitated by HPLC. 

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