ABSTRACT
Limitations .................................................................................................... 136 8.2.1 Enzyme-Linked Immunosorbent Assays and Radioimmunoassays ..... 136 8.2.2 Biochemical Techniques Based on Cell Fractionation and
Coimmunoprecipitation .................................................................... 137 8.2.3 Analysis of Substrate Phosphorylation ............................................. 137
8.3 Electrophysiological and Biophysical Techniques ........................................ 138 8.3.1 Cyclic Nucleotide-Gated Channels as Sensors ................................ 138
8.3.1.1 Advantages ......................................................................... 138 8.3.1.2 Requirements and Limitations ........................................... 138
8.3.2 Targeted FRET Biosensors ............................................................... 139 8.3.2.1 Advantages ......................................................................... 139 8.3.2.2 Requirements and Limitations ........................................... 139
8.4 Development of Targeted FRET Biosensors ................................................ 139 8.4.1 Design and Cloning .......................................................................... 139 8.4.2 Testing the Proper Sensor Localization ............................................ 141
8.4.2.1 Cell Culture and Transfection Materials ........................... 141 8.4.2.2 Cell Culture and Transfection ............................................ 141 8.4.2.3 Confocal Microscopy ......................................................... 142
8.4.3 FRET Measurements in Live Cells .................................................. 142 8.4.3.1 Materials and Instrumentation ........................................... 142 8.4.3.2 FRET Microscopy ............................................................. 143 8.4.3.3 Ofine Data Analysis ........................................................ 144
8.5 Discussion and Future Perspectives .............................................................. 144 Acknowledgments .................................................................................................. 145 References .............................................................................................................. 145
Cyclic nucleotides 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are ubiquitous second messengers that regulate myriads of physiological functions ranging from memory formation to vasorelaxation and cardiac contractility (Beavo and Brunton 2002; Hofmann and Wegener 2013).
