Retinas from postnatal day 15 (P15) rats were dissected and
homogenized by sonication in 5 mM Tris–acetate buffer with 65 mM NaCl,
2 mM MgCl2, and protease inhibitors. Proteins
were solubilized by a 4-hour incubation with 2% digitonin at 4°C and
deglycosylated by an overnight incubation at room temperature with
N-glycosidase F.
Western blot analysis with several serial protein dilutions was
performed to locate a concentration within the linear range of
detection. Protein in solution with sample dye, with B-ME and sodium
dodecyl sulfate for denaturation, was loaded and separated
electrophoretically on a 12% polyacrylamide gel. Protein in the gel
was then wet-transferred overnight to a polyvinylidene fluoride
membrane (Bio-Rad, Hercules, CA). The membrane was blocked, probed with
the N-terminal rhodopsin antibody rho4D2, washed, and then probed with
horseradish peroxidase–conjugated secondary antibody (Sigma, St.
Louis, MO). Labeling was detected with a Renaissance enhanced
chemiluminescence system (NEN Life Science Products, Boston, MA) and
hyperfilm-ECL X-ray film (Amersham Life Science, Arlington Heights,
IL). Densitometric analysis was performed on NIH image software
(available at rsb.info.nih.gov/nih-image). Analysis of relative levels
of protein expression was performed on samples with 5 μg of protein
per lane. Results from two lanes from each of 4 rats were averaged
before relative levels of expression were compared.