19] Purification of rat retinal rod outer segment membranes

Abstract

This chapter discusses the purification of rat retinal rod outer segment (ROS) membranes. The retinal ROS membrane is a highly specialized unit capable of detecting a single photon in visual transduction. ROS preparations obtained from bovine and frog retinas are widely described and used. Rats are easily reared under controlled conditions of diet and environmental light, important variables for the visual system. To maintain rhodopsin in its dark-adapted state within the ROS membrane, it is necessary to perform the entire preparation procedure under dim red light. When ROS rhodopsin is maintained fully dark adapted in this way, the final purity and total yield obtained from the preparations are easily estimated using rhodopsin's absorption spectrum. The initial portion of this method is based on an extensive modification of the procedure described by Paper-master and Dreyer. The purity and yield of ROS membranes that have been harvested from either or both of bands one or two can be rapidly estimated spectrophotometrically.