Purifier, centrifugal sedimentation

The catalytic activity responsible for the conversion of p-coumarlc acid Into p-hydroxybenzolc acid was sedimentable at 15 OOO g and could be further purified by sedimentation velocity centrifugation. By means of marker enzymes It was demonstrated that mitochondria are the organelles housing the respective enzymes, and that the mitochondrial Inner membrane is the site where the formation of p-hydroxybenzolc acid and then, most probably, further conversion into ubiquinone takes place (Table 1). 

Preparation of Organo Hectorites. Hectorite SHCa-1 was obtained from the Qay Mineral Society Repository at the University of Missouri, Columbia. The mineral was purified by sedimentation and subsequent treatment with NaHSO solution to remove carbonates. A 1.0 wt. % aqueous suspension of sodium hectorite (100 mL, 0.73 meq) was mixed with a 0.073 M solution of the chloride or bromide salt of the desired onium ion (20 mL, 1.46 mmol) dissolved in either water or ethanol. The products were then washed free of excess onium salt with ethanol as determined by a bromide or chloride specific electrode. The clay was then resuspended in de-ionized water, centrifuged and air dried at room temperature. 

Drugs have been purified by SPE in the analysis of amphetamine (AM) by Kaleta et al. [98], by various consecutive washing steps with hexane in the analysis of methamphetamine (MA) by Jones-Lepp and Stevens [99], and by simple centrifugation after addition of water, to separate the aqueous extract from a bottom sediment layer and a top fat layer, in the analysis of AM, MA, cocaine (CO), and benzoylecgonine (BE) by Langford et al. [100], who found little improvement in reducing matrix effects when applying SPE cleanup. 

The pyrogenic flame hydrolyzed silica Aerosil 200, a commercial product from Degussa, was used as a dispersion in doubly distilled water (1). The precipitated silica was prepared by hydrolysis of orthosilicic acid tetraethylester in ammoniacal solution according to the method of Stober, Fink and Bohn (11). The prepared suspension was purified by repeated centrifugation, separation from solvent and redispersion of the sediment in fresh water. Finally, the water was evaporated and the wet silica dried at 150°C for about half an hour. 

Isolation of human neutrophils. Leukocytes were obtained from normal donors by leukapheresis with an IBM 2997 Blood Cell Separator (8). Normal mature neutrophils were purified from this mixed leukocyte preparation by dextran sedimentation and Ficoll-Hypaque gradient centrifugation as previously described (9). The Wright-stained smears of the preparation showed that the cells were over 95% neutrophils. 

Centrifuge 17,000g for 30 min at 4°C when cellular DNA sediments along with SDS. The supernatant contains 80% of the viral DNA which can be further purified by CsCl centrifugation. 

The purified enzymes from A. niger and R. delemar contain some 13% of carbohydrate, and were shown by density-gradient ultra-centrifugation to be glycoproteins. Sedimentation and diffusion measurements indicated a molecular weight of 100,000 for both enzymes. ... 

Procec/Mre. - - Determinations on a micro scale are performed in 3-ml conical Pyrex centrifuge tubes. A volume of antiserum (containing antibody or myeloma antibody or crude seed extract or purified lectin) previously centrifuged until it no longer deposits sediment and containing about 6-8 fig of antibody N (AbN) or lectin N in a volume of about 50-100 fil is added to tubes containing a suitable range of accurately mea-... 

Preparation of H,K-ATPase enriched microsomal vesicles. H,K-ATPase-containing gastric microsomal vesicles were isolated from rabbit stomach as previously described (12). Crude microsomes were harvested from homogenized mucosa of unstimulated rabbit stomach (H2 receptor-blocked) as the membrane pellet sedimenting between 10 min at 13,000 x g and 1 hr at 100,000 x g. The pellet was resuspended in 10% sucrose, brought to 40% sucrose (9 ml), and overlaid with successive layers of 30% sucrose (11 ml), 10% sucrose (16 ml) [300 mM sucrose, 5 mM tris(hydroxymethyl)aminomethane (Tris), and 0.2 mM EDTA, pH 7.4] in a 37 ml tube. After centrifugation at 80,000 x g for 4 hr, the purified gastric microsomal vesicles were collected from the interface between 10% and 30 % sucrose and stored at 4° C until use. 

A technique called differential centrifugation is commonly used to fractionate particles into pellet and supernatant. The pellet is an aggregate of all sedimenting components, while the supernatant is a purified portion of the sample containing only the slowest sedimenting components. In this way, differential centrifugation is a purification technique in which large particles are removed from the supernatant. 

Dopa, dopamine, and tyrosine have been the most common substrates in the preparation of synthetic eumelanins. In a typical experiment the enzyme (15 ml of solution of 30 mg of enzyme in 100 ml Sorensen s buffer, pH 7) and L-dopa (150 mg) in pH 7 buffer (500 ml) are kept with access to air for 2 weeks (755) stirring, bubbling air through the mixture, and raising the temperature up to 38°C accelerate the process. Melanin is separated by filtration, fractional sedimentation, or, more efficiently, by centrifuging (500 to 100,000 g), especially after acidification (pH < 3.5) (see Section IV) (251). Samples are further purified by repeated resuspension and centrifugation or dialysis (252). 

In some cases it is possible to purify conjugates by density gradient centrifugation, provided that the buoyant densities or sedimentation coefficients are sufficiently different (Mannik and Downey, 1973 Ford et al., 1978). 

In Chapter 3, we considered the principles of centrifugation and the uses of centrifugation techniques for separating proteins and nucleic acids. Similar approaches are used for separating and purifying the various organelles, which differ In both size and density and thus undergo sedimentation at different rates. 

The liquid which contains the cells and non-assimilated hydrocarbons is divided by centrifuging into three streams cell paste, water phase and hydrocarbon phase. A part of the aqueous phase is recycled. The sediment can be treated again with O2 and nutrient solution in a purifying fermenter and oxidized once more. Then we must centrifuge... 

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