Sucrose density gradient centrifugation fractions collections

For calculation it was assumed that these five phospholipids together accounted for all of the phospholipids of the membrane. In the actual analyses, other phospholipids (primarily lysophosphatidyl choline and lysophosphatidyl ethanolamine) were found to account for less than 5% of the total. The heavy and light microlipid droplet fractions were obtained by sucrose density gradient centrifugation. Heavy fractions were collected between 0.5 and 1.0 M sucrose and light fractions banded at the 0.5 M sucrose-buffer interface. 

Following such treatment, the cultured cells in monolayer are washed with phosphate-buffered saline and extracted in 4 ml 0.5% Triton X-100 in saline/EDTA (100 mM NaCl, 10 mM EDTA, pH 8.0) for 2 min at room temperature. This releases most of the cytoplasmic material whilst the nuclei remain attached to the culture dish. 0.5% sodium dodecyl sulphate and 40 //g/ml pancreatic RNAse (preincubated at 80°C for 10 min, to inactivate DNAse) in saline/EDTA (above) is then added and the mixture incubated for 20 min at 37°C. One volume of chloroform/isoamyl alcohol (20 5, v/v) is then added and the phases mixed gently. The aqueous phase is separated by centrifugation and extracted again with chloroform/isoamyl alcohol. DNA is precipitated from the aqueous phase with 2 vol 95% ethanol and resuspended in 0.01 M Tris, pH 7.5. Alkaline sucrose density gradients (5-20%) are prepared in 0.1 M NaCl, 0.1 M NaOH with a final volume of 4.1 ml. Samples of DNA (max 3 fig) are layered on the top of these gradients and spun at 32 000 rpm at 20°C in a SW.50.1 Beckman rotor for 120 min. Fractions are collected and the [3H]DNA precipitated... 

Fig. 9. Sucrose density gradient pattern of carboxylase inactivated with [y- P]ATP. Partially purified carboxylase was incubated with 0.2 mM cyclic AMP for 30 minutes. Enzyme was then inactivated in the presence of 2 mM citrate and 1 mM ATP or 1 mM [y.32p]ATP (28 /xCiZ/ttmole) for 6 minutes. Inactivation was terminated by the addition of EDTA to a concentration of 50 mM. The sucrose density gradient contained 2 mM citrate and centrifugation was carried out as described previously except that the period of centrifugation was 135 minutes. Twenty-four fractions were collected. Aliquots were assayed for carboxylase activity with 10 mM citrate and the location of radioactive...

These results clearly indicate that the bulk of the in vitro sialic acid incorporation into endogenous gangliosides and sialo-glycoproteins was in retina membranes other than ROS. Centrifugation of ROS free membranes in sucrose density gradients resulted in the isolation of the more active membranes for the transfer of H-NeuNAc from CMP- H-NeuNAc, which were collected at the junction between the sucrose layers of densities 1.10 - 1.11. The labelled lipids and proteins found in the ROS membranes after they were incubated with CMP- H-NeuNAc could arise from Pla membranes contaminating the crude ROS fraction during fractionation of retinas. 

Fio. 3. Subcellular localization of endogenous NA and tritiated NA( H-NA) in rat vas deferens. After the administration of H-NA in vivo, the tBsue was homogenized and layered onto a sucrose density gradient for centrifugation. Appearance of the tube after centrifugation is indicated on the left. A hole was made in the bottom and the contents collected in five-drop fracticms. Alternate fractions were assayed for H-NA and for endogenous NA. Both n re distributed in the supernatant fluid and in a fraction of small vesicles just below the supernatant, which also contained microsomal particles. (Reproduced with permission frcnn Potter, L. T. and Axelrod, J., J. Pharmac. Exp. Ther., 142, 291-298, 1963.)... 

Fig. 5. RNA of plastid fraction (washed 1000g pellet) isolated from leaves maintained in darkness or illuminated for 3 hours after absorption of P-phosphate. RNA was extracted and centrifuged as described in legend for Fig. 3, but fractions here were collected manually and optical densities at 260 m/ were determined after dilution. (The two heavier RNA s, when obtained from maize chloro-plasts purified on a sucrose density gradient, are approximately 22 S and 17 S. The apparent base composition of these RNA s is approximately 25% adenine, 32% guanine, 20% uracil, and 23% cytosine these values are based on the distribution of radioactivity, after paper electrophoresis, among AMP, GMP, UMP, and CMP obtained by alkaline hydrolysis of plastid RNA s prepared from sucrose gradient purified plastids of maize leaves supplied P-phosphate.)...

Samples (0.3 ml) of the labeled extracts were centrifuged through a linear 5-20% sucrose density-gradient (in 0.01 M sodium acetate and 0.1 M NaCl at pH 5.0) at 37,000 rpm for 5 hours at 5-10°C. Three-drop (about 0.20 ml) fractions were collected after bottom puncture of the centrifuge tube. These were diluted with an equal volume of distilled water and the 260 m/i absorption was determined in each fraction. Measurements of radioactivity were then made on the. same samples by scintillation counting. From Piatigorsky and Tyler (1967). 

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