Biology Reference
In-Depth Information
CHAPTER
11
Monitoring Receptor
Oligomerization by
Line-Scan Fluorescence
Cross-Correlation
Spectroscopy
Mark A. Hink and Marten Postma
Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy (LCAM),
Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, The Netherlands
CHAPTER OUTLINE
Introduction ............................................................................................................ 198
11.1 Materials........................................................................................................199
11.1.1 Solutions, DNA Constructs, and Cells ........................................... 199
11.1.2 Fluorescent Proteins ................................................................... 200
11.1.3 Microscope ................................................................................ 200
11.2 Methods .........................................................................................................202
11.2.1 Growth and Transfection of HeLa Cells ......................................... 202
11.2.2 Microscope Calibration ............................................................... 203
11.2.3 Fluorescence Fluctuation Measurements ...................................... 204
11.2.4 Auto- and Cross-Correlation Analysis ............................................ 205
11.3 Discussion......................................................................................................209
Acknowledgments ................................................................................................... 210
References ............................................................................................................. 210
Abstract
Membrane-localized receptor proteins are involved in many signaling cascades, and
diffusion and oligomerization are key processes controlling their activity. In order to
study these processes in living cells, fluorescence fluctuation spectroscopy tech-
niques have been developed that allow the quantification of concentration levels, dif-
fusion rates, and interactions between fluorescently labeled receptor proteins at the
nanomolar concentration level. This chapter presents a brief introduction to the tech-
nique and a protocol to measure and quantify the diffusion and oligomerization of
 
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