Ultracentrifugation analytical

The cells are formed from centrepieces which are sandwiched between two solid quartz discs, forming an absorption cuvette. 

In principle, there are two forms of analytical ultracentrifuge experiment sedimentation equilibrium and sedimentation velocity. These can provide different, and to some extent complementary, information about the samples under study (Byron 1996). 

It is not straightforward to determine the position of the moving boundary. Even with a single species, the boundary is broadened by diffusion, making accurate 

The initially homogeneous solution forms a boundary which moves progressively from left to right, becoming broader as it does so as a result of diffusion. The plateau level decreases with time as a consequence of the sector shape of the cell. 

The occurrence of two bands can be readily explained when the complex, which is in a dynamic equilibrium, dissociates then the small and large molecules sediment together for a short period before re-forming the faster sedimenting complex. The duration of the dissociation process is generally a few seconds, which is short compared with the duration of the sedimentation run. From the concentrations of the species in the two bands, the equilibrium constant of the interaction can be evaluated. 

The maximum density of the gradient usually does not exceed the density of the particles. Because of this, if the centrifugation continues indefinitely, the particles pellet at the tube bottom. The gradient is chosen to have the minimal density and viscosity possible to allow rapid separation and minimize diffusional band broadening. 

In rate-zonal centrifugation the conditions for sensitive biological particles are gentle (low osmotic pressure, g forces, and viscosity) and typically the separation is faster than isopycnic separations. Applications include the separation of proteins and nucleoproteins, like ribosomes. 

The isopycnic method has been used to dramatically demonstrate semiconservative DNA replication, using CsCl density gradients. Separation of DNA, RNA, protein, and carbohydrates can be performed in dense CsCl solutions, where the RNA pellets, the DNA forms bands, and protein and carbohydrates form a thin layer called a pellicle at the top of the gradient. 

Centrifugation techniques are used mainly in separation and purification protocols, or as a sample preparation step in analytical biochemistry. Historically, analytical 

The sample is poured into a cavity in a single or double sector centerpiece, made of an aluminum alloy or an epoxy resin material [Fig. 13.9(h)]. The centerpiece is fitted with transparent windows made of quartz, or sapphire if high g forces are required. A cell housing (with related parts including spacers, gaskets and screw rings) that holds the cell assembly fits into the ultracentrifuge rotor. A simplified 

The (outward) force exerted on an object of mass m, rotating at a distance r from the axis with angular velocity o, is equal to mro/. This is equivalent to an acceleration of ro/. 

Q What is the effective acceleration (in g) at a radius of 10 cm in a centrifuge rotor spinning at 15,000 rpm  

The angular volcoly is the ate of rotation in radians per second One complete rotation (360 ) is equal to 2it radians 

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Apart from tliese mainstream metliods enabling one to gain a comprehensive and detailed stmctural picture of proteins, which may or may not be in tlieir native state, tliere is a wide variety of otlier metliods capable of yielding detailed infonnation on one particular stmctural aspect, or comprehensive but lower resolution infonnation while keeping tlie protein in its native environment. One of tlie earliest of such metliods, which has recently undergone a notable renaissance, is analytical ultracentrifugation [24], which can yield infonnation on molecular mass and hence subunit composition and their association/dissociation equilibria (via sedimentation equilibrium experiments), and on molecular shape (via sedimentation velocity experiments), albeit only at solution concentrations of at least a few tentlis of a gram per litre. 

Schuster T M and Toedt J M 1996 New revolutions in the evolution of analytical ultracentrifugation Curr. Opinion Ceii. Bioi. 6 650-8... 

FIGURE 9.23 Analysis of ultrahigh poly(acrylamjde). MW 48 million by analytical ultracentrifugation. Eluent 0.1 M Na2SO<. Flow rate 0.3 ml/min. Columns PSS Suprema 30000, 20 /tim, 8 x 300 mm. Oven temp 30°C. Detector Rl. Standards PSS polyacrylamide standards. 

Analytical ultracentrifugation (AUC) Molecular weight M, molecular weight distribution, g(M) vs. M, polydispersity, sedimentation coefficient, s, and distribution, g(s) vs. s solution conformation and flexibility. Interaction complex formation phenomena. Molecular charge No columns or membranes required [2]... 

Harding SE, Rowe AJ, Horton JC (eds) (1992) Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK... 

Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society... 

Minton AP (1994) In Schuster TM, Laue TM (eds) Modern Analytical Ultracentrifugation. Birkhauser, Boston, 81... 

With this relationship for all samples was calculated from ninh This M is used for evaluating the reaction data. The ultracen rifuge (u.c measurements were carried out in a Spinco model E analytical ultracentrifuge, with 0.4% solutions in 90% formic acid containing 2.3 M KCl. By means of the sedimenta- ion diffusion equilibrium method of Scholte (13) we determine M, M and M. The buoyancy factor (1- vd = -0.086) necessary for tSe calculation of these molecular weights from ultracentrifugation data was measured by means of a PEER DMA/50 digital density meter. 

Techniques which seem less suitable for routine size analysis are (1) analytical ultracentrifugation combined with a Schlieren optical system (Mason and Huang, 1978 Weder and Zumbuehl, 1984) (2) the sedimentation field flow fractionation (SFFF) technique to separate heterogeneous dispersions (e.g., Kirkland et al., 1982). 

Techniques for study of higher orders of protein stmcture include x-ray crystallography, NMR spectroscopy, analytical ultracentrifugation, gel filtration, and gel electrophoresis. 

The membrane-bound preparation from kidney is easily solubilized in non-ionic detergent and analytical ultracentrifugation shows that the preparation consists predominantly (80 85%) of soluble af units with 143000 [28]. The soluble a)S unit maintains full Na,K-ATPase activity, and can undergo the cation or nucleotide induced conformational transitions that are observed in the membrane-bound preparation. A cavity for occlusion of 2K or 3Na ions can be demonstrated within the structure of the soluble a)S unit [29], as an indication that the cation pathway is organized in a pore through the aji unit rather than in the interphase between subunits in an oligomer. 

Hensley P. Defining the structure and stability of macromolecular assemblies in solution the re-emergence of analytical ultracentrifugation as a practical tool. Structure 1996 4 367-373. 

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