Preparative ultracentrifugation

Ultracentrifugation Preparative Density gradient Hydrated density... 

The sedimentation boundary of an enzyme preparation in an aqueous buffer at 20.6 C was measured after various times in an ultracentrifuge at 56,050 rpm. The following results were obtained ... 

Each interferon preparation was ultracentrifuged at 20,000 revolutions per minute for one hour to remove tissue debris and inactivated virus. The supernatant was dialyzed against distilled water (1 400) for 24 hours at4°C. The material was then freeze-dried. The dried product was reconstituted in one-tenth of the original volume in distilled water and dispensed into ampoules. Reconstituted solutions were assayed for interferon activity, examined for toxicity, and tested for sterility. 

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. 

Bar = 50 nm. Samples were prepared at the initial concentrations of 2 raM lipid, 0.22 mM SQDG and 8pg Chla/ml PS II and concentrated by ultracentrifugation in the presence of Mes-Hepes/betaine buffer (pH 7.0). 

The usefulness of the ultracentrifuge in the preparation of samples rather than in the production of analytic data should not be overlooked. Preparative ultracentrifuges have utility in fractionating polymer samples and in freeing them from easily sedimented impurities. 

There are various methods for the determination of the size distribution of organic pigment particles, the most common are sedimentation techniques in ultracentrifuges and specialized disk centrifuges as well as electron microscopy. These methods require considerable experimental skill, since the results depend largely on sample preparation and especially on the quality of the dispersion. 

Procedure 3,3 Preparation of sub-cellular fractions using ultracentrifugation... 

Svedberg, T. Pederson, K.O. (1940). The Ultracentrifuge. Oxford University Press. Tiselius, A. (1947). Adsorption analysis of amino-acids. Adv. Protein Chem. 3, 67-93. Young, E.G. (1963). Occurrence, Classification, Preparation and Analysis of Proteins. In Comprehensive Biochemistry (Florkin, M. Stotz, E.H., Eds.), Vol. 7, pp. 1-55. Elsevier, Amsterdam. 

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