The CRAC Channel

This volume discusses the cellular and molecular techniques used to study and characterize the different components of the CRAC channel signaling pathway. The chapters in this book cover topics such as fluorescence-based measurements of the CRAC channel activity in cell populations; patch-clamp recording of the CRAC channel in STIM-Orai overexpressing cells and native systems; western-blotting and co-immunoprecipitation of endogenous STIM/ORAI and protein partners; shRNA-mediated gene silencing; and the SC-SMD system. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Thorough and cutting-edge, The CRAC Channel: Methods and Protocols is a valuable resource for any researcher interested in learningmore about the CRAC channel, as its activation mechanism and roles in cellular functions have yet to be completely explored.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt
Patch-Clamp Recording of the CRAC Channel Current in STIM-Orai Overexpressing Cells.- Fluorescence-Based Ratiometric Measurement of CRAC Channel Activity in STIM-Orai Overexpressing HEK-293 Cells.- Recording SOCE Activity in Neurons by Patch-Clamp Electrophysiology and Microfluorometric Calcium Imaging.- Mn2+ Quenching Assay for Store-Operated Calcium Entry.- Fluorescence-Based Measurements of Store-Operated Ca2+ Entry in Cancer Cells using Fluo-4 and Confocal Live Cell Imaging.- Fluorescence-Based Measurements of the CRAC Channel Activity in Cell Populations.- Indirect Measurement of CRAC Channel Activity using NFAT Nuclear Translocation by Flow Cytometry in Jurkat Cells.- CRAC Channel Components Quantitative Expression (In Tissues and Cell Lines) using qPCR.- Western-Blotting and Co-Immunoprecipitation of Endogenous STIM/ORAI and Protein Partners.- Study of Endogenous CRAC Channels in Human Mast Cells using an Adenoviral Delivery System to Transduce Cells with Orai-Targeting shRNAs or with cDNAs Expressing Dominant-Negative Orai Channel Mutations.- Store-Operated Ca2+ Entry in Drosophila Primary Neuron Cultures.- Study of the Endogenous CRAC Channel using shRNA-Mediated Gene Silencing.- Engineered Cross-Linking to Study the Pore Architecture of the CRAC Channel.- Measurement of the CRAC Channel Fast Ca2+-Dependent Inactivation (FCDI).- High Resolution Imaging of STIM/Orai Subcellular Localization using Array Confocal Laser Scanning Microscopy.- Single Channel Single Molecule Detection (SC-SMD) System.