Biology Reference
In-Depth Information
Detergent
micelle
2
SCHEME 15.1
Single-molecule immunoprecipitation (pull-down) for single-molecule detection (SMD)
fluorescence experiments. We developed the following scheme to self-assemble the depicted
structure. The glass surface of the chip is first coated with glycidoxy propyl silane (GOPS) to
introduce reactive epoxy groups on the glass surface. Biotinylated BSA is then covalently
attached to the surface by overnight reaction with the epoxy groups. The biotinylated
BSAefficiently capturesNeutrAvidin (A), adeglycosylated formof the tetrameric biotin-binding
protein avidin with a more neutral isoelectric point, and NeutrAvidin captures a biotinylated
antibody (1D4 mAb). 1D4 mAb that recognizes the C-terminal nonapeptide of rhodopsin
(“C9-tag”), which we routinely use as purification tags for other GPCRs. The remaining biotin
binding sites are saturated with biotin to convert the avidin tetramer in the high-affinity
statewith fMbinding affinity for biotin. The antibody captures theAlexadye-labeledGPCRby its
C-terminal C9-tag (green) from detergent solution.
region allows long-term observation of a single molecule (
Scheme 15.1
). In this way,
slow dissociation/association processes can be observed (
Huber & Sakmar, 2011
).
15.2.3
Automated, multicolor SMD-TIRF microscopy
The number of single-molecule fluorescence techniques is large and steadily grow-
ing, but for the study of GPCR signalosomes, a smaller number of methods appear to
be especially well suited. They are based on (i) intensity and residence time of
