Rhodopsin Extraction from Mouse Eyes

Mouse eyes were enucleated in darkness under dim red light. Each eye was flash-frozen on dry ice immediately after enucleation. Rhodopsin was extracted with 20 mm HEPES, pH 7.4, containing 10 mm n-dodecyl-β-maltoside and 5 mm freshly neutralized NH₂OH·HCl, as described previously (Palczewska et al., 2018 (link)). Briefly, the tissue was homogenized with 0.9 ml of buffer in a 2-ml Dounce tissue homogenizer (Kontes Glass Co) and shaken for 5 min at 4°C. The sample was then centrifuged at 17,200 × g for 5 min at 4°C. The supernatant was collected, the pellet was extracted a second time with 0.9 ml of buffer, and the combined supernatants were filtered through a 0.22-μm polyethersulfone membrane. Absorbance spectra were recorded using a Varian Cary 50-Scan UV-Vis spectrophotometer (Varian Australian Pty Ltd.); the sample was used as blank, then it was bleached for 5 min with a white-light, 875-Lumens bulb, and finally the difference absorbance spectrum was recorded immediately following a bleach. The concentration of rhodopsin was determined by the decrease in absorbance at 500 nm using the molar extinction coefficient ε_500nm = 42,000 M⁻¹ ·cm⁻¹.