Figure 13. In vitro Formation of the Rhodopsin-G_t Complex.

Purified G_t and purified rhodopsin in 0.5 mM DDM were mixed in equimolar amounts. The sample was light activated, incubated under ambient light for 20 minutes, and analyzed by gel filtration chromatography on a Superdex 200 10/300 GL size exclusion column equilibrated in 50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM MgCl₂, 1 mM EDTA, and 0.5 mM DDM. A. The rhodopsin:G_t complex elutes as a homogeneous, Gaussian peak at 11.5 mL. B. Fractions collected across the peak in (A) analyzed on a 4–12% gradient polyacrylamide gel (Invitrogen). Rhodopsin, Gα_t, and Gβ1 are observed.