Discontinuous density gradient

Chung, B.H. (1980) Preparative and quantitative isolation of plasma lipoproteins rapid single discontinuous density gradient ultracentrifugation in a vertical rotor. 

E. Labeling of Cells for Light Microscope Stiidles, The procedure for labeling cells with microspheres larger than O.h jj, was identical to that described above except (l) a low speed centrifuge co ild be used, and (2) the cells could be separated from unreacted particles by means of a discontinuous density gradient. The cells remained at the interface of the gradient and the particles sedimented to the bottom of the container. 

Gl. Goldrosen, M. H., Gannon, P. J., Lutz, M., and Holyoke, E. D., Isolation of human peripheral blood lymphocytes Modification of a double discontinuous density gradient of Ficoll-Hypague. ]. Immunol. Methods 14, 15-17 (1977). 

Fig. 3. Distribution of non-occluded ( ) and occluded ill ID ChAc in fractions separated by discontinuous density gradient centrifuging. The blocks correspond to the fractions 0-1 described by Whittaker et at. (1964). A, Suspension of hypo-osmotically treated synaptosomes and B, After binding of soluble ChAc to synaptosome membranes. (Reproduced from Biochem. J. (1968), F.

P4. Piomelli, S., Lurinsky, G., and Wassennan, L. R., The mechanism of red cell aging. 1. Relationship between cell age and specific gravity evaluated by ultracentrifugation in a discontinuous density gradient. J. Lab. Clin. Med. 69, 659 (1967). 

Several methods have been employed to isolate and purify type II cells, including enzymatic digestion of pulmonary tissue to free lung cells from the pulmonary epithehum and purification of type II pneumocytes by discontinuous density gradients (Kikkawa and Yoneda 1974), primary culture (Dobbs and Mason 1979), centrifugal elutriation (Jones et al. 1982), or unit gravity sedimentation (Brown et al. 1984). 

Endoplasmic reticulum. Hypocotyls of 4-days-oId dark-grown soybean were harvested and incubated overnight with [l-I C]acetate. A fraction enriched in ER was isolated by discontinuous density gradient centrifugation followed by aqueous polymer two-phase partition [4]. 

Sphaeroplasts were prepared by slight modifications to published methods [12,13]. Lysis of sphaeroplasts was effected by a combination of osmotic lysis and gentle mechanical disruption [14]. Discontinuous sucrose-density gradients were constructed and fractions were then assayed for protein, PG and marker enzymes for different organelles. 

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

When discontinuous sucrose gradient centrifugation is used, two different density sucrose solutions should be piled up into the tube. If 20% (w/w) and 30% (w/w) density solutions are piled up, the ER-enriched fraction should be layered on the interphase between 20% (w/w) and 30% (w/w) sucrose solutions. 

Method Density gradient. Rate-zonal. The rate-zonal method is one of six addressed by SpinPro. The other methods are differential, differential-flotation, discontinuous, isopycnic, and 2-step isopycnic. These methods differ dramatically in their set up, principles of operation, and expected results. The rate-zonal method is described here briefly so that the recommendations to follow can be appreciated. Prior to the run in a rate-zonal method, a gradient material is introduced to the rotor tubes in steps of increasing density from the top to the bottom of the tube. The sample to be separated is layered, as a thin band, on the top of the gradient. As the run begins, each component in the sample moves toward the bottom of the tube. Some components sediment faster than others. This fact is the basis for the separation. If the run parameters are appropriate, the components will form separate bands within the gradient. At the conclusion of the run, the band representing the component of interest can be removed from the tube. 

Density gradients maybe divided into three types preformed discontinuous gradients (e.g.. Protocol 5.3.1), preformed continuous gradients (e.g.. Protocol 5.3.2) or self-generating gradients (e.g.. Protocol 5.3.3). Materials used for density gradients are classified as ionic and non-ionic media (Table 5.1). 

Glycosphingolipid Glycosyltransferase Assays. A 25-33% (vol/vol) homogenate of mouse tumors or harvested cells in 0.32 M sucrose containing 0.1% 2-mer-captoethanol and 0.001 M EDTA (pH 7.0) was used as enzyme source. Membrane fractions for glycolipid gly-cosyltransferase assays were isolated at the junction of 0.32 M and 1.2 M on a discontinuous sucrose density gradient (32,43). 

The ab initio approach includes all a- and 71-electrons and seeks to use either analytical or numerical solutions to the integrals that occur in the quantum mechanical problem. This procedure was initially carried out within the framework of the one electron HF-SCF method using the basis sets described above. Subsequently it has been implemented using density functional theory (DFT). If the electron density in the ground state of the system is known, then in principle this knowledge can be used to determine the physical properties of the system. For instance, the locations of the nuclei are revealed by discontinuities in the electron density gradient, while the integral of the density is directly related to the number of electrons present. Ab initio methods are obviously computationally intensive. 

Either the post-mitochondrial or the microsomal fraction is taken for further purification by continuous [63] or discontinuous [62,64] density-gradient centrifugation. A fraction thus obtained is often used as enzyme preparation. This fraction has a density of 1.10-1.15 and the (K -I-H )-ATPase in this fraction can usually be stimulated by K -specific ionophores (valinomycin, nigericin). 

Isolation of LDL and Measurement of the Inhibition of LDL Oxidation. Blood samples were obtained from food-deprived cholesterol-fed rabbits with or without drug treatment. Plasma lipoproteins were isolated by discontinuous NaCl/KBr density gradient ultracentrifugation in a SW 50 Ti rotor (Beckman Instruments, Palo Alto, CA) for 20 h at 4°C. LDL was isolated in a density range of 1.019-1.063 g/mL, and was dialyzed extensively at 4 C under Nj against PBS (5 mM phosphate and 145 mM NaCl, pH 7.4) in darkness. LDL cholesterol was determined enzymatically. All samples were diluted in normal saline to give a final concentration of 150 mg/dL cholesterol for the oxidation susceptibility studies. 

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