Biology Reference
In-Depth Information
CHAPTER
17
Detection of G Protein-
Coupled Receptor (GPCR)
Dimerization by
Coimmunoprecipitation
Kamila Skieterska
1
, Jolien Duchou
1
,B´atrice Lintermans, and Kathleen Van
Craenenbroeck
Laboratory of Eukaryotic Gene Expression and Signal Transduction (LEGEST),
Ghent University-UGent, Ghent, Belgium
CHAPTER OUTLINE
Introduction ............................................................................................................ 324
Specific Interactions with ER Chaperones ............................................................... 324
Dimerization........................................................................................................... 325
Interaction with “Gatekeepers” ............................................................................... 325
17.1 Materials........................................................................................................326
17.2 Methods .........................................................................................................327
17.2.1 Overexpression of GPCRs in Mammalian Cell Lines ....................... 327
17.2.2 Cell Lysis................................................................................... 327
17.2.2.1 Mechanical Disruption ....................................................... 327
17.2.2.2 Nondenaturing Detergent-Based Lysis Methods.................. 329
17.2.2.3 Denaturing-Based Lysis Methods: Urea Lysis
and SDS-Lysis ................................................................................. 332
17.2.3 Coimmunoprecipitation............................................................... 332
17.2.3.1 Standard Coimmunoprecipitation: in Total Cell Lysate ........ 333
17.2.3.2 Membrane Coimmunoprecipitation: on Living Cells ............ 335
17.2.4 Elution ...................................................................................... 335
17.2.5 Detection of Interacting Partners ................................................. 337
17.2.6 Controls..................................................................................... 337
17.3 Discussion......................................................................................................338
Acknowledgments ................................................................................................... 339
References ............................................................................................................. 339
1
Both authors contributed equally to this work.
