7 Preparing Rod Outer Segment Membranes, Regenerating Rhodopsin, and Determining Rhodopsin Concentration

This chapter discusses simple and rapid techniques for the preparation of bovine rod outer segments (ROSs), determination of the concentration of rhodopsin, and the regeneration of rhodopsin. These procedures require access only to a refrigerated centrifuge and a spectrophotometer in a dark room. They lend themselves well to modifications to fit existing equipment or desired experimental protocols. The chapter also discusses the methods that are used for measuring the amount of rhodopsin present and reconstituting rhodopsin from opsin. ROSs are easy to prepare owing to their unusually low density compared to other retinal cells and cell organelles. The chapter also explains the method that uses the discontinuous sucrose density gradient procedure. This procedure and variations of it yield outer segments containing variable amounts of soluble ROS proteins. However, the procedure of discontinuous gradients is found to be preferable as it is easier and faster to prepare and a good yield of rhodopsin from either fresh or frozen retinas (0.5–0.8 mg/retina) with acceptable levels of soluble ROS proteins is obtained.