Thesis

Tissue fractionations in reorienting density gradients

In view of their greatly increased sample capacity and optimized internal geometry, zonal rotors have become increasingly popular instruments for quantitative centrifugal fractionations of animal and plant tissues on density gradients. Nearly all zonal rotors presently in use require a rotating- seal assembly which necessitates dynamic loading and unloading of the rotor. As a consequence of certain disadvantages which accompany dynamic unloading and numerous difficulties encountered in the use of a rotating seal, the possibility of employing simpler, seal-less zonal rotors to effect similar quantitative biological separations was investigated. Three seal-less zonal rotors were tested which were loaded either statically or dynamically but were always statically unloaded. The rotors were designed so that the liquid density gradient underwent a stable reorientation between a vertical and radial array during acceleration and deceleration. Rotor performance was evaluated using variety of techniques and the rotors then applied to a series of quantitative tissue fractionations. These included: (l) the separation of whole homogenates of liver into fractions containing nuclei, mitochondria, lysosomes, rnicrosomes and soluble constituents; (2) the separation of discrete populations of liver nuclei on the basis of size differences; (3) the separation of lysosomes and peroxisomes using a replenished gradient technique; (4) the isolation of protein bodies from seeds and (5) the separation of intact and broken chloroplasts from plant leaves. In addition to providing a simpler means for separating cellular components by zonal centrifugation, the reorienting gradient rotors were found to provide a number of interesting and important advantages over zonal rotors with rotating seals.

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