Isolation and Fractionation of Retinal Components

Frozen bovine retinas were thawed and gently vortexed in buffer A (50% sucrose, 10 mM HEPES, pH7.4, 1 mM EDTA, 5 mM MgCl2) supplemented with a protease inhibitor cocktail (Complete; Roche, Mannheim, Germany). After 13,000g centrifugation (1 hour), rod outer segments (ROS) were collected and the pelleted “rest of retina” (ROR) resuspended in Buffer A without sucrose. A centrifugation step (750g, 10 minutes) isolated nuclei. The ROR was centrifuged (100,000g, 1 hour) to separate soluble proteins (ROR-S1) from membranes (ROR-P1). The ROR-P1 was loaded on a 1.2 M sucrose cushion and centrifuged (200,000g, 30 minutes). Synaptosomes (Syn-S2) were collected from the top of the sucrose cushion; pelleted material was the “rest of membranes” fraction (ROM-P2). Dilutions for antibodies to retinal markers were: anti-RDS (1:30, Per 2B6, gift from Robert Molday); mouse anti-NKA (1:1000, M7-PB-E9, Santa Cruz Biotechnology, Inc.); rabbit anti-HCN1 (1:2500)^{33 (link)}; mouse anti-actin (1:1000, AC-74, Sigma-Aldrich Corp., St. Louis, MO, USA); rabbit a-GAPDH (1:250, Abcam); rabbit anti-RAB3 (1:500, Thermo Fisher Scientific, Inc.); rabbit anti-VAMP2 (Synaptic Systems, Göttingen, Germany), mouse anti-PSD-95 (1:500, Millipore, Billerica, MA, USA). Rat brains were collected and fractionated according to the previously described protocol.^{34 (link)}