Ab8227

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.

Samples for western blotting were collected in 2X homemade Laemmli buffer [82 ], boiled for 10 minutes, cooled and loaded onto 10% pre-cast gels (Bio-Rad) for separation by SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Thermo Fisher Scientific) using wet transfer (100V for 90 minutes on ice) and membranes were blocked using 5% milk (Morrisons, Edinburgh, UK) for at least 30 minutes. Membranes were washed three times with TBS-T (Tris-buffered-saline/0.1% Tween 20 (Sigma)) and incubated overnight at 4°C with primary antibodies in 2% BSA/TBS-T (0.1%). Commercial antibodies were used for β-tubulin (clone YL1/2, Bio-Rad, Watford, UK; 1:5000), β-actin (Abcam ab8227; 1:2000), ZAP (Invitrogen PA5-31650; 1:1000), TRIM25 (Abcam ab167154; 1:1000) and KHNYN (Insight Bio sc-514168; 1:1000). In-house rabbit polyclonal antibodies raised against NP and PB2 are previously described [83 (link)] and were both diluted 1:1000. Membranes were then washed 3 times and incubated with species-specific secondary antibodies conjugated to Alexafluor-680 or -800 (Thermo Fisher Scientific) diluted 1:5000 in 2% BSA/TBS-T (0.1%) for 90 minutes. Membranes were washed 3 times and visualised using a LICOR Odyssey Fc imaging system.