Biology Reference
In-Depth Information
CHAPTER
Homo-FRET 16
Cecilia de Heus * , Nivard Kagie { , Raimond Heukers * ,
Paul M.P. van Bergen en Henegouwen * , and Hans C. Gerritsen {
* Cell Biology, Department of Biology, Science Faculty, Utrecht University, Utrecht, Netherlands
{ Molecular Biophysics, Department of Soft Condensed Matter and Biophysics, Science Faculty,
Utrecht University, Utrecht, Netherlands
Analysis of EGF Receptor
Oligomerization by
CHAPTER OUTLINE
Introduction ............................................................................................................ 306
16.1 Theory Homo-FRET Quantification ....................................................................309
16.1.1 Steady-State Fluorescence Anisotropy Imaging ............................. 309
16.1.2 Time-Resolved Fluorescence Anisotropy Imaging........................... 311
16.2 Materials........................................................................................................313
16.2.1 Plasmid Constructs..................................................................... 313
16.2.2 Cell Lines .................................................................................. 315
16.2.3 Microscope ................................................................................ 315
16.2.3.1 Confocal Time-Resolved and Steady-State Fluorescence
Anisotropy Imaging Microscopy ........................................................315
16.3 Methods .........................................................................................................316
16.3.1 Slide Preparation........................................................................ 316
16.3.1.1 Reference Measurements ...................................................316
16.3.1.2 Predimerization Measurements ...........................................316
16.3.1.3 Ligand-induced Oligomerization ..........................................317
16.3.2 Analysis..................................................................................... 317
16.4 Discussion......................................................................................................317
Acknowledgments ................................................................................................... 319
References ............................................................................................................. 320
Abstract
Growth factor receptors are present in the plasma membrane of resting cells as mono-
mers or (pre)dimers. Ligand binding results in higher-order oligomerization of
ligand-receptor complexes. To study the regulation of receptor clustering, several
 
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