To begin, enucleate the eye globes of the euthanized mouse and homogenize the eyes in an ice cold buffer. Centrifuge the homogenate at 16, 000 G for 15 minutes at four degrees Celsius. Discard the supernatant containing water soluble proteins and other contaminants.
Then resuspend and solubilize the pelleted membrane and membrane proteins in a buffer for one hour at four degrees Celsius on a rotating platform. Afterward, centrifuge the resolubilized membrane fraction for one hour at 16, 000 G at four degrees Celsius. Mix the supernatant with 30 to 50 microliters of Rho1D4 antibody beads, and blank immunoglobulin G antibody beads in two separate independent samples and incubate for one hour at four degrees Celsius on a rotating platform.
Using a magnetic stand, separate the pull-down by collecting the beads. Discard the supernatant and wash the beads with a high and low salt buffer containing Bis-tris propane, sodium chloride, and N-dodecyl-beta-D-maltoside. To elute the rhodopsin protein, incubate the beads for five to 10 minutes at room temperature with a 65 microliter buffer of the given composition.
Then using a rectangular sub-micro 50 microliter quartz cubit with a two millimeter aperture and 10 millimeter path length, analyze the eluate for absorbance from 200 nanometers to 800 nanometers in a fast scan setting with a spectrophotometer. To validate and determine the quality, expose the eluate to bright light for one minute. Then repeat the absorbance measurement.
Subtract the blank absorbance and calculate the ratio of the analyzed absorbent spectra. Then plot the blank subtracted absorbent spectra, and calculate the free opsin value. Afterward, using Beer-Lambert law, calculate the concentration of rhodopsin, then calculate the concentration of ligand-free opsin, and estimate the difference in concentration for apo-opsin, and ligand-free opsin in amount and percentage.
