Biology Reference
In-Depth Information
15.7.4 Step 4: Immobilization of NeutrAvidin ....................................... 291
15.7.4.1 Materials ......................................................................... 291
15.7.4.2 Solutions and Buffers....................................................... 291
15.7.4.3 Procedure ....................................................................... 292
15.7.5 Step 5: Immobilization of Biotinylated Antibody.......................... 292
15.7.5.1 Materials ......................................................................... 292
15.7.5.2 Solutions and Buffers....................................................... 292
15.7.5.3 Procedure ....................................................................... 293
15.8 Protocol 6: Automated Multicolor, Single-Molecule TIRF Microscopy .................293
15.8.1 Step 1: Microscope Optical Setup.............................................. 295
15.8.2 Step 2: Alignment of Laser-to-Fiber, Fiber-to-Fiber Beam
Combination System................................................................. 298
15.8.3 Step 3: Alignment of DualView and QuadView Simultaneous
Imaging Systems ...................................................................... 299
15.8.4 Step 4: Calibration of the Multiwell Plate Geometry..................... 299
15.8.5 Step 5: Adjust TIRF Excitation .................................................. 300
15.8.6 Step 6: Sample for Subpixel Alignment of Color Channels............ 300
15.8.7 Step 7: Alignment of the CRISP Focus-Stabilization (autofocus)
System .................................................................................... 300
15.8.8 Step 8: Preparation of Antibleaching and Antiblinking Imaging
Buffer System.......................................................................... 300
15.8.8.1 Materials ......................................................................... 300
15.8.8.2 Solutions and Buffers....................................................... 301
15.8.8.3 Procedure ....................................................................... 301
15.8.9 Step 9: Multiposition, Multicolor, Time-Lapse Imaging of Single
Molecules ................................................................................ 302
15.8.10 Step 10: Image Processing for Optimal Detection of Single
Molecules..................................................................................... 302
Acknowledgments ................................................................................................... 302
References ............................................................................................................. 302
Abstract
Heptahelical G protein-coupled receptors (GPCRs) mediate transmembrane signal
transduction to facilitate intercellular communication. GPCRs assemble in the mem-
brane bilayer with a variety of cytoplasmic adapter and scaffold proteins to formmo-
lecular machines, or “signalosomes,” which undergo complex dynamic assembly and
disassembly reactions. Despite significant recent advances in structural studies of GPCRs
and their associated cytoplasmic components, understanding transmembrane signaling in
four dimensions with chemical precision requires new approaches. One promising ap-
proach to study allosteric effects involved in signalosome reaction pathways is to usemul-
ticolor single-molecule detection (SMD) fluorescence experiments in biochemically
defined systems. We describe here the methodological foundation for automated,
