Protein reader

Total cellular protein was extracted by M-PER mammalian protein extraction reagent buffer (Pierce, 78501) with proteinase inhibitor (Roche Diagnostics), and quantified by Bio-Rad protein reader. Protein samples (20 μg) were then separated on 10% Tris–Cl gradient gel and electro-blotted onto nitrocellulose membrane. The membranes were incubated in blocking buffer for 1 hr at room temperature, washed three times in PBS with 0.1% Tween for 5 min each, and incubated with primary antibody in blocking buffer overnight at 4°C. Primary antibodies against the following proteins were used for western blots: RPE65 (1:1,000 Novus Biologicals, NB100-355), BESTROPHIN-1 (1:500 Novus Biologicals, NB300-164), CRALBP (1:500 Abcam, ab15051), β-actin (1:2,000 Abcam, ab8227), and GFP (1:5,000 Invitrogen, A6455). Mouse and rabbit secondary antibodies were obtained from Santa Cruz and used at a concentration of 1: 5000.

Cell pellets were extracted by the M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, 78501) supplemented with proteinase inhibitors (Roche, 04693159001), and the protein concentration was quantified by a Bio-Rad protein reader. After denaturing at 95°C for 5 min, the samples (20 μg) were run on 4–15% gradient SDS-PAGE gel at room temperature, and wet transferred onto nitrocellulose membrane at 4°C. The membranes were incubated in blocking buffer containing 5% (w/v) non-fat milk for 1 hr at room temperature and subsequently incubated overnight at 4°C in blocking buffer supplemented with primary antibody. Primary antibodies against the following proteins were used: CRALBP (1:500 Abcam, ab15051), RPE65 (1:1,000 Novus Biologicals, NB100-355), β-Actin (1:2,000 Abcam, ab8227), BEST1 (1:500 Novus Biologicals, NB300-164), His (1:1,000 Fisher Scientific, PA1983B), and Myc (1:1,000 Fisher Scientific, PA1981). Fluorophore-conjugated mouse and rabbit secondary antibodies (LI-COR Biosciences, 925–68070 and 925–32213, respectively) were used at a concentration of 1:10,000 and an incubation time of 1 hr at room temperature, followed by infrared imaging.