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. Author manuscript; available in PMC: 2015 Jan 14.
Published in final edited form as: Biochemistry. 2013 Dec 20;53(1):127–134. doi: 10.1021/bi4012995

Figure 1.

Figure 1

Preparation of rhodopsin-containing Nanodiscs. The N2C,E113Q,D282C rhodopsin triple mutant was reconstituted with 11-cis-retinal, inserted into the lipid bilayer of Nanodiscs, and purified by immunoaffinity chromatography on 1D4-Sepharose. Dark, Absorption spectrum of the rhodopsin-containing Nanodiscs purified in the dark at pH 7.5; light activated, spectrum following 2 min illumination with light from a 300w tungsten bulb; acid trapped, spectrum immediately following the addition of 0.5% SDS and 50 mM sodium phosphate buffer, pH 3.5, to the light exposed sample. The acid trapped spectrum has been corrected for the effect of dilution. Inset, SDS-PAGE analysis of the steps used for preparation and purification of the rhodopsin containing Nanodiscs. Lane 1, rhodopsin-MSP1D1 mixture following detergent removal but before loading onto the 1D4-Sepharose column; lane 2, unbound material from the 1D4-Sepharose column; lane 3, last wash before elution; and lane 4, 1D4-peptide eluate containing purified N2C,E113Q,D282C rhodopsin triple mutant in MSP1D1 Nanodiscs. Protein bands in the gel were visualized with Coomassie Blue.