Cross-Linking of Cone Opsins in Membranes

HEK-293 cells were transiently transfected with WT green, red, green-IAM, and red-TSV constructs cloned into the pcDNA3.1(+) vector. Forty-eight hours after transfection, cells from two 10 cm plates were harvested, washed with PBS, and suspended in 200 μL of 20 mM Bis-Tris-propane, 100 mM NaCl buffer (pH 7.5) containing a protease inhibitor cocktail. After gentle trituration with a syringe and needle, the membranous fraction was pelleted by centrifugation at 16000g for 10 min at 4 °C. The pellet was suspended in 200 μL of the Bis-Tris-propane buffer, and half of the resulting solution was used for cross-linking. Two millimolar disuccinimidyl glutarate (DSG) cross-linker (Thermo Scientific, Waltham, MA) was added to membranous samples, and the cross-linking reaction was allowed to proceed for 2 h on ice. The reaction was terminated with 1 M Tris-HCl (pH 8.0), added to a final concentration of 50 mM, and the samples were incubated for 15 min followed by membrane solubilization with 20 mM DDM. Cross-linked opsins (50 μg of total protein extract) were separated on a 10% SDS-PAGE gel and then transferred to polyvinyl difluoride (PVDF) membranes. Proteins were detected with an anti-rhodopsin C-terminal 1D4 antibody, an HRP-conjugated anti-immunoglobulin, and a chemiluminescence assay (Thermo Scientific). The amount of opsin dimer was quantified by densitometry using ImageJ.