Resolution of components from rat liver homogenate in reorienting density gradients

Abstract

The subcellular components of rat liver homogenate were separated into seven distinct components when discontinuous gradients were used. The studies reported here demonstrate the similarity of the separation when compared with the work of previous investigators (4). From rest to slow acceleration or deceleration to 3,000 rpm, there is very little sedimentation of the macromolecules in the sample. The additional time required to accelerate the reograd rotor when compared to the dynamically loaded rotor does not pose a problem. The hub of the core in the reograd rotor is much larger than in the dynamically loaded rotor; hence the sample is placed in a higher centrifugal field during the initial acceleration. This reduces the dilution or smearing of the sample during the slow acceleration process. Additional sedimentation of the macromolecules due to the lengthy acceleration and deceleration times can be calculated effectively based on the rotor speed versus the time. A specially designed reorienting gradient core for the zonal rotor was used to permit static loading and unloading. This technique is quite stable for isolating components from plant and animal tissue homogenates. Furthermore, the reograd technique can also be applied to the isopycnic or rate zone separations of selected ribosomes, starch, and especially DNA strands since the sample material is not subjected to the shearing forces of a rotating seal assembly (1).