Our research investigates how vitamin A homeostasis is regulated, focusing on physiological interaction between retinal binding protein receptor 2, RBPR2, and its ligand, RBP4, for whole-body vitamin A transport. We also explore how 11-cis-retinal binds to rhodopsin, which is crucial for phototransduction and vision. We use two known techniques to establish a link between vitamin A storage and distribution by liver receptors, and the impact of interrupting this mechanism in the wellness of vision by accumulating disease-causing lipofuscins.
Our protocol utilizes two techniques, focusing on distant communication between the liver and the eye for vitamin A transport. Our protocol makes it more evident and easier to correlate the disease conditions caused by dietary or hereditary issues. The blood serum protein and factors stabilize the communication between the retinol binding protein receptor and its ligand, RBP4.
Finding all the relevant mechanisms is complicated, yet essential for the next part in answering some unexplored areas. In the future, our focus will be on translating the research protocol, studying the correlation of disease phenotypes in human patient samples, and looking for genetic variants of vitamin A receptors in disease. Begin by immobilizing the purified msRBP4 protein on the sensor chip.
To do so, go to the Run menu, choose the Wizard, and proceed with the Immobilization option. Next, dilute the ligand in immobilization buffer to a concentration of 10 micrograms per milliliter, and aliquot the coupling reagents into vials. Now, open the Immobilization Setup Wizard window.
Select the CM5 chip and Immobilize flow cell 2 options. Then set the target level to 1, 200 response units for an R maximum of 30 response units in the kinetic study. Choose ethanolamine as the wash solution.
Enter the name RBP4 and click Next. In the Rack Position dialogue box, click Eject Rack. Remove the rack.
Place the vials containing the specified volumes of each reagent, and then insert the rack with the sample vials. After that, click Ok, and Next. Finally, in the Prepare Run Protocol dialog click Start, and then Save.
To set up the kinetic assay for RBPR2 and STRA6 peptides, serially dilute the mouse RBPR2, mutant RBPR2, and STRA6 peptides in running buffer. Open the control software and select the Kinetics/affinity wizard. In the Injection Sequence dialogue, select Flow path 2-1, with Flow cell 2 as active and Flow cell 1 as reference.
In the Setup dialogue, check the Run Startup Cycles option. Input the solution as buffer, specify the number of cycles, and click Next. Now, go to the Injection Parameters dialogue and enter sample parameters such as contact time of 120 seconds, flow rate of 30 microliters per minute, and dissociation time of 300 seconds.
For regeneration parameters, set the regeneration solution to glycine hydrochloric acid, with a contact time of 30 seconds and a flow rate of 30 microliters per minute. Then, click Next. In the Samples dialogue, enter the peptides names, molecular weights, and concentrations.
Then, click Next. In the Rack Position dialogue, assign the diluted peptides as individual 120 microliter samples with various concentrations from 1 to 100 micromolar to the designated positions on the rack. Click Eject Rack, and insert all the vials.
Afterward, click Next. And then, in the Prepare Run Protocol dialogue, click Start. Enter a file name and click Save.
Now, in the software, click the Start menu and select Evaluation. Open the file, then from the dropdown selection menu, choose FC 2-1 to analyze the reference cell blank-subtracted active sensorgram for the association and dissociation phases of msRBP4, mutant msRBPR2, and msSTRA6 peptides. Next, click the dropdown Kinetics/affinity button and choose Surface Bound.
In the Select Curves dialogue, check Include Column in the Table and click Next. Now, go to the Select Data dialogue, view the blank-subtracted sensorgrams. Click Kinetics and Affinity, keep the default parameters, and click Fit for curve fitting.
Inspect the Report tab for equilibrium dissociation constant KD, association rate KA or kon, and dissociation rate KD or koff. Save the kinetics data sensorgram in tab-delimited text format, and use the values to plot graphs. 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 4 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 4 degrees Celsius on a rotating platform.
Afterward, centrifuge the re-solubilized membrane fraction for one hour at 16, 000 g at 4 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 4 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 5 to 10 minutes at room temperature with a 65 microliter buffer of the given composition. Then, using a rectangular sub-micro 50-microliter quartz cuvette with a 2 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 absorbance spectra.
Then, plot the blank-subtracted absorbance 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.
