Cellular components, centrifugation

Conditions for the Separation of Selected Cellular Components by Centrifugation... 

Fig. 5. Separation in Latham bowl (a) whole blood is pumped down the feed tube and enters bowl at bottom (b) centrifugal force spins denser cellular components outside, leaving plasma or platelet-rich plasma (PRP) in inner band (c) when bowl is full, plasma flows out effluent tube, followed by platelets and then leukocytes, until bowl is almost completely full of ted cells (d) after draw is completed, bowl stops spinning and uncoUected components are...

Centrifugation can be used either as a preparative technique for separating and purifying macromolecules and cellular components or as an analytical technique to characterize the hydrodynamic properties of macromolecules such as proteins and nucleic acids. 

Separation into components can only be achieved by stopping the process when sedimentation of the desired component has occurred. The sediment is then resuspended in fresh solvent and centrifuged at a lower speed, when the heavier particles will sediment leaving the component in suspension. Such a method is known as differential sedimentation and is particularly useful for the fractionation of cellular components. The method outlined in Procedure 3.3 is simple and is designed to separate four main cellular fractions, namely, nuclear, mitochondrial, microsomal and soluble. 

Nuclei already sediment at low accelerations that can be achieved with bench-top centrifuges. Decanting the residue (the supernatant ) and carefully suspending the sediment (or pellet ) in an isotonic medium yields a fraction that is enriched with nuclei. However, this fraction may still contain other cellular components as contaminants—e.g., fragments of the cytoskeleton. 

In this chapter we will explore the underlying principles of centrifugation and discuss the application of this technique to the isolation and characterization of biological molecules and cellular components. 

The specimen, as drawn, contains cells, platelets, fibrin, and particulates. For most chemistry tests, e.g., determination of glucose, cholesterol, etc, it is necessary to first separate the cellular blood fraction which, if present during the assay, would interfere with the determination and adversely affect the accuracy of the measurement. The cellular components arc separated by centrifugation. 

Blood is drawn as whole blood, that is, cells and plasma. Drug concentrations are measured in plasma, not whole blood. Whole blood is spun down in a centrifuge to allow separation of plasma from the cellular components. Plasma is more homogeneous than whole blood and therefore easier to analyze. The blood-to-plasma ratio of most drugs falls in a range of 0.5-1.5, and a value of 1 is generally not an unsafe assumption. In other words, the concentration of a drug in whole blood is often assumed to be the same as that... 

After fermentation, the yeast cells were harvested and broken mechanically in a bead mill, and differential centrifugation was used to partition the cellular components into water-soluble and insoluble fractions. SDS-polyacrylamide gel electrophoresis in conjunction with Western blot analysis 36) revealed that the polyphenolic protein aggregated with the insoluble cellular protein. 

Sedimentation coefficients are usually expressed in Svedberg units (S), equal to lO g. The smaller the S value, the slower a molecule moves in a centrifugal field. The S values for a number of biomolecules and cellular components are listed in Table 4.2 and Figure 4.14. 

The separation of the components of the myelin-free crude mitochondrial fraction of whole brain tissue in centrifuge tubes is compared with a separation by zonal centrifugation. On a shallow, step-wise gradient of 0.8-1.7 M sucrose in a BXIV rotor of 650 ml capacity, it was possible to obtain lysosomal, mitochondrial, synaptosomal and plasma membrane fractions after spinning for 2 h at 67,000 x g. These fractions were characterised by enzyme markers and other means. At least two synaptosomal populations could be clearly separated, one of which could actively take up a-methylnoradrenaline. Some preliminary studies on the uptake of [ A/e-14C]-choline into sub-cellular components after intracerebral injection are also described. 

Equilibrium density-gradient centrifugation, which separates cellular components according to their densities, can further purify cell fractions obtained by differential centrifugation. 

Starch isolation from tubers/roots involves washing and peeling the tubers. The tubers are soaked in an aqueous solution of sodium bisulfite to prevent discoloration. The tubers are ground by a cylindrical drum containing rotary blades. Starch is isolated from other cellular components by centrifugation. 

As described above, the formation of xanthan-protein complexes induced by salts such as calcium or magnesium chloride depends strongly on the quality of the solution. It was previously reported that when xanthan solutions contained less impurities, interactions between macromolecules and calcium ions were less important, and that the pH limit corresponding to precipitation was shifted to basic pH (75). Here, when xanthan C broth was centrifuged, thixotropic phenomena were less apparent. So, for this xanthan solution, it is clearly shown that the presence of cellular components was essential to induce interactions between xanthan chains and cations. It can also be noted that the xanthan broth was frozen before all the experiments and thus, that the cells may have been lysed thereby releasing all their constituents inside the medium. Because of the diversity and complexity of the cellular components, it is difficult to predict which kind of interactions can be involved in the aggregation. 

Cesium chloride is used to prepare dense solutions required for isolating cellular components with a centrifuge. A 40.0 wt% solution of CsCl (FM 168.36) has a density of 1.43 g/mL. 

Homogenization of Tissues 3 Differential Centrifugation Technique 3 Separation of Cellular Components Intracellular Distribution of Biochemical Components 7 Distribution of Basic Cellular Constituents Reference Compounds Distribution of Multiple-Enzyme Systems... 

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