Biology Reference
In-Depth Information
CHAPTER
6
Biochemical and Imaging
Methods to Study Receptor
Membrane Organization and
Association with Lipid Rafts
Bruno M. Castro
*
, Juan A. Torreno-Pi ˜ a
*
, Thomas S. van Zanten
*
, and
Maria F. Gracia-Parajo
*
,{
*
ICFO—Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels
(Barcelona), Spain
{
ICREA—Institucio´ Catalana de Recerca i Estudis Avan¸ats, Barcelona, Spain
CHAPTER OUTLINE
Introduction and Rationale....................................................................................... 106
6.1 Methods to Determine Protein Association with Lipid Rafts................................. 107
6.1.1 Lipid Raft Disruption by Cholesterol-Depleting Agents ....................... 107
6.1.1.1 Methyl-b-Cyclodextrin .............................................................. 108
6.1.1.2 Cholesterol Oxidase ................................................................. 108
6.1.2 Colocalization with Putative Lipid Raft Markers................................. 109
6.2 Optical Techniques to Study Receptor Membrane Organization and
Association with Lipid Rafts.............................................................................. 110
6.2.1 Confocal Fluorescence Microscopy ................................................... 110
6.2.2 Homo-F ¨ rster Resonance Energy Transfer Microscopy........................ 110
6.2.3 Superresolution Techniques to Map the Nanolandscape of the
Cell Membrane.............................................................................. 114
6.2.3.1 Far-Field Localization Techniques (STORM/PALM) ................... 114
6.2.3.2 Far-Field Patterned Illumination Techniques (STED) ................. 115
6.2.3.3 Near-Field Techniques (NSOM) ............................................... 117
Concluding Remarks ............................................................................................... 118
Acknowledgments ................................................................................................... 119
References ............................................................................................................. 119
