FIGURE 7.

Quantitation of Bio-DOPA¹ cross-linking of Ste2p Lys²⁶⁹ mutant receptors by immunoblot analysis. Cross-linked membrane samples were divided into two parts; one part was mixed with His-Select^TM HC-nickel affinity gel (H) to enrich Ste2p receptors, and the other part was mixed with avidin affinity beads (A) to enrich only cross-linked receptors. The samples eluted from the His affinity and avidin affinity beads were resolved on 10% SDS-PAGE and probed with anti-FLAG antibody to detect Ste2p. The band intensities were quantitated using a Chemi-Doc XRS photodocumentation system, and the percentage of cross-linking was calculated as described under “Experimental Procedures.” H, nickel affinity-purified receptors; A, avidin affinity-purified receptors.