Biology Reference
In-Depth Information
CHAPTER
14
Analysis of Receptor-
Receptor Interaction by
Combined Application of
FRET and Microscopy
Sonal Prasad, Andre Zeug, and Evgeni Ponimaskin
Cellular Neurophysiology, Center of Physiology, Hannover Medical School, Hannover, Germany
CHAPTER OUTLINE
Introduction and Rationale....................................................................................... 244
14.1 Theory.......................................................................................................... 245
14.1.1 Basic Principles of FRET........................................................... 245
14.1.2 Fluorescent Proteins and FRET.................................................. 247
14.1.3 Principle Behind Lux-FRET Quantification and its Outcome ......... 248
14.2 Materials / Experimental Setups..................................................................... 249
14.2.1 Reagents ................................................................................. 249
14.2.2 Devices ................................................................................... 251
14.2.3 Reagent Setup ......................................................................... 252
14.2.3.1 Preparation of Cell Culture Media..................................... 252
14.2.3.2 Buffer A for Cell Lysis ...................................................... 252
14.2.3.3 Buffer B for FRET Imaging and Fluorolog Experiments ..... 252
14.2.3.4 Protease Inhibitors and Phosphate-Buffered Saline Stock
Solutions ........................................................................................ 253
14.3 Methods (Protocols and Procedure) ............................................................... 253
14.3.1 Cell Preparation and Transfection Timing ................................... 253
14.3.1.1 Culture of Cells ................................................................ 253
14.3.1.2 Reculture and Seeding Cells ............................................ 253
14.3.1.3 Transfection .................................................................... 253
14.3.2 Cell Lysis for Fluorolog.............................................................. 254
14.3.3 Procedure Part I: Data Acquisition ............................................. 254
14.3.3.1 Setting Individual Protocols According to
Experimental Setup ........................................................................ 255
14.3.4 Spectroscopic Measurement at a Fluorescence
Spectrometer (Fluorolog 3v.2.2.) ............................................... 255
