For immunoprecipitation, antibodies to opticin (dilution, 1:50) and chicken GH (dilution, 1:1000) were used, corresponding to 1 to 5 μg antibody protein. The antibody was mixed with 100 μL (200–500 μg protein) of vitreous in immunoprecipitation buffer, (in 0.5% sodium dodecyl sulfate, 1.0 mM sodium orthovanadate, 10 mM Tris buffer [pH 7.4], for denaturing conditions) with 400 μL water and 500 μL of 2× immunoprecipitation buffer. The mixture was vortexed and incubated at 4°C for 1 hour. Protein A-Sepharose (50 μL; Amersham Biosciences, Montréal, Québec, Canada) was added, vortexed, and incubated overnight with agitation. After centrifugation (16,000g for 4 minutes) and two washes in immunoprecipitation buffer, the pellet was resuspended in 30 μL sample buffer containing β-mercaptoethanol, boiled for 5 minutes, and centrifuged again.
For polyacrylamide electrophoresis, 30 μL of supernatant was added to each well of a 8% gel, and run for 48 minutes at 140 V. Retina lysates were prepared in protease-inhibitor buffer, containing 15 μg/mL aprotinin, 1 μg/mL leupeptin, 5 μg/mL pepstatin, and 1.74 mg/mL phenylmethylsulfonyl fluoride (PMSF), and 20 μg of this lysate protein in 2× sample buffer containing bromophenol blue and β-mercaptoethanol was loaded in each lane. Samples were transferred from the gels onto a supported nitrocellulose membrane at 100 V for 2 hours. For immunoblot analysis, membrane blocking was performed with 5% skimmed milk in Tris-buffered saline with 0.1% Tween 20. Primary antibodies were incubated for 18 hours at 4°C followed by four washes for 10 minutes each. For staining GH bands, biotinylated goat secondary antibodies (1:100 dilution; Sigma-Aldrich Co., St. Louis, MO) were used for 1.5 hours at room temperature (RT). Proteins were peroxidase labeled (Vectastain ABC Reagent; Vector Laboratories Inc., Burlington, Ontario) for 1.5 hours at RT. For staining opticin bands, peroxidase-conjugated goat secondary antibodies were used at a dilution of 1:100. In both cases, the chemiluminescence reaction (ECL; Amersham Biosciences) was used for color development. Negative controls were performed using the primary antibody after preabsorption with antigen.
Preabsorption of anti-GH antibody was performed by mixing 1 mg/mL recombinant chicken GH with the antiserum at a dilution of 1:3000 (Amgen, Thousand Oaks, CA). Preabsorption of the anti-opticin antibody was performed by mixing equal parts of the antibody with the peptide antigen used to raise the antibody.