Centrifugation preparative models

Carrots (Boleo) were peeled by 2% NaOH at 88-96°C for 4 minutes, minced by a meat mincer (2 mm) and homogenised for 2 minutes by an ULTRA-TURRAX T25 homogenizer (from Jahne Kunkel). The carrot mash was preheated to 45°C (20 minutes) before the enzyme preparations, 25 mg enzyme protein/kg mash, were added. The enzymes were dissolved in water to give a dilution of 5% (v/v) of the carrot mash. The mash was incubated at 45°C under stirring (60 rpm) for 2 hours, before the enzymes were inactivated at 86°C for 5 minutes in a microwave oven. Finally the purees were homogenised for 1 minute by ULTRA TURRAX. The viscosity of the puree was measured by a BROOKFIELD viscosimeter Model DV-n + with spindle A from HELIPATH SPINDLE SET at 2.5 rpm thermosta-ted at 50°C. The stability of the puree was measured as the sediment (in %) after centrifugation in 10 ml tube a 1660 x g for 10 minutes. 

Fig. 2. Preparation of linear sucrose density gradient, centrifugation, and fractionation. Fractionation is performed using an ISCO model 640 density gradient fractionator in this experiment.

Liposomes (SUVs) were prepared by probe sonication according to standard procedures (31) in the presence of STPP. A mixture of lecithin, cholesterol, and STPP (PC/Ch/STPP = 65/15/20, molar ratio final total lipid 25 mg/ mL) was dissolved in chloroform followed by removal of the organic solvent using a rotary evaporator. After adding 5 mM HEPES (pH 7.4) to the dry lipid film, the sample was probe sonicated with a Sonic Dismembrator (Model 100, Fischer Scientific) at a power output of approximately 10 W for 30 minutes. To remove any titanium particles, which have been shed from the tip of the probe during sonication, the sample was centrifuged for 10 minutes at 3000 X g. The formed liposomes were separated from free, i.e., nonincorporated, STPP by gel filtration chromatography on a Sephadex G-15 column. 

Preparation and analysis of SDS extracts from digester sludge. The particulates from a 30-ml sample were removed by centrifugation (15,00Qg) at 4 C for 20 min. The particulates were washed three times with 100 mM Tris buffer pH 7.0 and resuspended in 15 ml of buffer. The extraction procedure consisted of agitating the sample with a Fisher model 346 rotator at 25 C in the presence of 0.1% SDS for 1 h. The particulate material was then removed by centrifugation at 15,000 g at 4 C for 20 min, and the supernatant was used to perform the enzyme assays. 

DuPont Instruments Sorvall (USA), Model RC-5B centrifuge, equipped with a Model SS-34 rotor for crude extract preparation. 

Sampling and Measurements. The determination of dissolved actinide concentration was started a week after the preparation of solutions and continued periodically for several months until the solubility equilibrium in each solution was attained. Some solutions, in which the solubilities of americium or plutonium were relatively high, were spectrophotometrically analyzed to ascertain the chemical state of dissolved species. For each sample, 0.2 to 1.0 mL of solution was filtered with a Millex-22 syringe filter (0.22 pm pore size) and the actinide concentration determined in a liquid scintillation counter. After filtration with a Millex-22, randomly chosen sample solutions were further filtered with various ultrafilters of different pore sizes in order to determine if different types of filtration would affect the measured concentration. The chemical stability of dissolved species was examined with respect to sorption on surfaces of experimental vials and of filters. The experiment was performed as follows the solution filtered by a Millex-22 was put into a polyethylene vial, stored one day, filtered with a new filter of the same pore size and put into another polyethylene vial. This procedure was repeated twice with two new polyethylene vials and the activities of filtrates were compared. The ultrafiltration was carried out by centrifugation with an appropriate filter holder. The results show that the dissolved species in solution after filtration with Millex-22 (0.22 ym) do not sorb on surfaces of experimental materials and that the actinide concentration is not appreciably changed with decreasing pore size of ultrafilters. The pore size of a filter is estimated from its given Dalton number on the basis of a hardsphere model used in the previous work (20). 

The homogeneity and molecular size of the purified antibody preparations were determined by ultracentrifiigation in a Spinco Model E centrifuge. The results of this analysis on anti-lactose antibodies are illustrated in the photographs of the ultracentrifuge patterns shown in Fig. (1). The... 

The antibody-coated red cells are prepared as previously described. It is particularly important for this procedure that the indicator red cells are absolutely free of clumps. The sensitivity of coated cells can be assayed by reverse passive hemagglutination if, as in the model under consideration, the antigen is available in soluble form. The cells under study are washed in suitable tissue culture medium or other buffered solution and suspended at a concentration of 10 per milliliter in the same diluent to which serum has been added (usually 1% fetal calf serum). A small volume (50-100 /U.1) of the cell suspension is placed in a 10 x 75 mm disposable tube. The addition of an equal volume of 1% coated red cells results in a mixture that contains about 25 red cells per lymphocyte. Linkage of antibody on the red cells to the corresponding antigen determinant on the surface of the lymphocyte results in the formation of a rosette or lymphocyte surrounded by red cells. The mixing of cells and incubation for at least 1 hr are done in an ice bath. The tubes are then centrifuged very briefly (1 min at 1000 g), and a drop of dye is added to tint the lymphocytes (e.g., crystal violet or brilliant cresyl blue). The mixture is then aspirated four or five times with a Pasteur pipette and examined in a hemacytometer chamber at about 400 x. A cell is scored as a rosette if it is surrounded by three or more adherent erythrocytes, and usually 300 cells are counted. 

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