[8] Preparation of retinal rod outer segments

This chapter discusses the preparation of retinal rod outer segments. Isolation of rod outer segments (ROS) and the assay of their purity must reflect the research goals that require their isolation. It is not sufficient to apply one procedure in rote fashion that was initially designed for one purpose and use the circular argument that the presence or absence of some component in the isolated membrane fraction is evidence for or against the existence of that component in the organelle in vivo. There are several factors to be carefully considered while modifiing each step: (1) the shearing forces employed during initial and subsequent homogenization; (2) the ionic strength, ionic composition, and osomolality of each of the media; (3) the presence or absence of reducing agents; (4) the presence or absence of nucleotide phosphates; (5) the possible addition of proteolytic inhibitors; and (6) the degree of light exposure of the retina and of isolated ROS.