Subcellular Calcium Measurements in Mammalian Cells Using Jellyfish Photoprotein Aequorin-based Probes

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2013 Oct 12

PMID 24113784

Citations 79

Authors

Massimo Bonora

Carlotta Giorgi

Angela Bononi

Saverio Marchi

Simone Patergnani

Alessandro Rimessi

Rosario Rizzuto

Paolo Pinton

Affiliations

Soon will be listed here.

Abstract

The jellyfish Aequorea victoria produces a 22-kDa protein named aequorin that has had an important role in the study of calcium (Ca(2+)) signaling. Aequorin reacts with Ca(2+) via oxidation of the prosthetic group, coelenterazine, which results in emission of light. This signal can be detected by using a special luminescence reader (called aequorinometer) or luminescence plate readers. Here we describe the main characteristics of aequorin as a Ca(2+) probe and how to measure Ca(2+) in different intracellular compartments of animal cells (cytosol, different mitochondrial districts, nucleus, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes and subplasma-membrane cytosol), ranging from single-well analyses to high-throughput screening by transfecting animal cells using DNA vectors carrying recombinant aequorin chimeras. The use of aequorin mutants and modified versions of coelenterazione increases the range of calcium concentrations that can be recorded. Cell culture and transfection takes ∼3 d. An experiment including signal calibration and the subsequent analyses will take ∼1 d.

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