Figure 6.
SDS/PAGE analysis of products formed in crosslinking of rhodopsin derivative S240C-R and Tαβγ. The photocrosslinking procedure was performed as described in Fig. 3. All samples were treated with 10 mM DTT immediately after the addition of Laemmli SDS sample buffer. The gel was visualized by Coomassie blue staining. (A) After GTP washing (Step 4) to remove uncrosslinked Tαβγ (lane 2), sample V was reduced by DTT and the eluted protein is shown in lane 1. Lane 3 contains 25 pmol of purified Tαβγ as a control. (B) Sample V in Fig. 3 was treated with NEM and eluted with DTT, and the eluate was then derivatized with MBB (Steps 5–7) in Fig. 3B. Sample VIII (Fig. 3B) then was analyzed by Western blotting using antibodies against Tα (I), Tβ (II), and biotinyl moiety (III). Lane 1 in all three cases contained purified Tαβγ as a control.