Biology Reference
In-Depth Information
CHAPTER
15
Site-Specific Labeling
of Genetically Encoded
Azido Groups for
Multicolor, Single-Molecule
Fluorescence Imaging
of GPCRs
He Tian, Thomas P. Sakmar, and Thomas Huber
Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York,
New York, USA
CHAPTER OUTLINE
15.1 Purpose..........................................................................................................270
15.2 Theory............................................................................................................270
15.2.1 Site-Specific Labeling of GPCRs with Fluorophores Suitable for
SMD-TIRF ............................................................................... 270
15.2.2 Single-Molecule Immunoprecipitation of GPCRs ......................... 271
15.2.3 Automated, Multicolor SMD-TIRF Microscopy............................. 272
15.3 Protocol 1: Genetically Encoded Azido Groups for Bioorthogonal Conjugation .....273
15.3.1 Step 1: Transfection of the HEK293-F Suspension Cells to Express
Azido-Group Tagged Opsin ........................................................ 273
15.3.1.1 Materials ......................................................................... 273
15.3.1.2 Culture HEK293-F Suspension Cells for Transfection ........ 274
15.3.1.3 Cotransfect the Cells with the Plasmids for Amber Codon
Suppression ................................................................................... 274
15.3.2 Step 2: Purification of Heterologously Expressed Wild-Type and
Azido-Tagged Rhodopsin .......................................................... 275
15.3.2.1 Materials ......................................................................... 275
15.3.2.2 Regeneration of Heterologously Expressed Opsin with
11-cis-retinal .................................................................................. 275
15.3.2.3 Purification of Rhodopsin................................................. 275
15.3.2.4 Characterization of Purified Rhodopsin with UV-Vis
Spectroscopy ................................................................................. 276
