It is now widely recognized, particularly for some of the larger organelles (nuclei, mitochondria, lysosomes, peroxisomes), which can be pelleted at relatively low g-forces (1000-15 000g), that the use of viscous, hyperosmotic sucrose gradients at g-forces of more than 100000g in long sedimentation path length swinging-bucket rotors, is both inconvenient and potentially deleterious to the organelles. Consequently, there is a pronounced trend to the use of low-viscosity gradient media such as Nycodenz® or iodixanol , whose gradients have a much lower osmolality than those of sucrose (or even be isoosmotic). Although the traditional swinging-bucket rotor is still widely used for density gradient centrifugation, the short sedimentation path length of vertical or near-vertical rotors not only reduces the centrifugation time, it also minimizes considerably the hydrostatic pressure imposed on the particles in the gradients. The new protocols in this chapter reflect these trends and concerns. The means by which discontinuous and continuous gradients are prepared and how they are harvested is too large a subject to be considered in this text; ref. 18 contains detailed accounts of these procedures.
Some protocols take the advantage of the ability of some of the modern density gradient media to form self-generated gradients. The colloidal silica particles in Percoll® can form such gradients at g-forces above 20000g, while other gradient media such as iodixanol, which is a true solute, requires g-forces in excess of 180000g. Because some of the colloidal silica particles will pellet during the centrifugation, vertical rotors (normally the most efficient rotors for such gradients) are not recommended for self-generated Percoll® gradients - fixed-angle rotors are more commonly used.
A disadvantage of Percoll® gradients is that it is normally a requirement to remove the colloidal silica particles prior to any analysis; the particles are light scattering and can interfere with any spectrophotometric measurements; they also cause irregular banding of proteins in SDS-PAGE. Moreover, removal of the silica colloid by sedimentation can also lead to the loss of organelles into the gel pellet that is formed . With the exception of spectrophotometry assays in the UV, if the organelle is at a sufficiently high concentration to permit direct analysis on the gradient harvest, then it is usually unnecessary to remove solutes such as sucrose, Nycodenz® or iodixanol. If solute removal is required then the organelles can simply be sedimented from the gradient harvest after dilution with two volumes of buffer, without loss of material .
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