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[Methods in Molecular Biology] Calcium Signalling Volume 1925 (Methods and Protocols) || High-Throughput Screening Using Photoluminescence Probe to Measure Intracellular Calcium Levels
摘要： Aequorin, a 22 kDa protein produced by the jellyfish Aequorea victoria, was the first probe used to measure Ca2+ concentrations ([Ca2+]) of specific intracellular organelles in intact cells. After the binding of Ca2+ to three high-affinity binding sites, an irreversible reaction occurs leading to the emission of photons that is proportional to [Ca2+]. While native aequorin is suitable for measuring cytosolic [Ca2+] after cell stimulation in a range from 0.5 to 10 μM, it cannot be used in organelles where [Ca2+] is much higher, such as in the lumen of endoplasmic/sarcoplasmic reticulum (ER/SR) and mitochondria. However, some modifications made on aequorin itself or on coelenterazine, its lipophilic prosthetic luminophore, and the addition of targeting sequences or the fusion with resident proteins allowed the specific organelle localization and the measurements of intra-organelle Ca2+ levels. In the last years, the development of multiwell plate readers has opened the possibility to perform aequorin-based high-throughput screenings and has overcome some limitation of the standard method. Here we present the procedure for expressing, targeting, and reconstituting aequorin in intact cells and for measuring Ca2+ in the bulk cytosol, mitochondria, and ER by a high-throughput screening system.
关键词： Cytosol,Calcium probes,Calcium,High-throughput screening,Aequorin,Mitochondria,ER
更新于2025-11-21 11:20:42
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Aequorin as a sensitive and selective reporter for detection of dopamine: A photoprotein inhibition assay approach
摘要： Dopamine is a metabolite that plays a key role in the human body and in biomedical and diagnostic applications. Thus, the concentration of this analyte has been considered in various diseases in therapeutic drug monitoring (TDM). In the present study, for the first time, a photoprotein inhibition assay strategy was developed by utilizing aequorin for the direct detection of dopamine as a receptor and reporter simultaneously. The results showed that bioluminescence emission of aequorin was effectively quenched by increasing concentration of dopamine at the range of 1 nM to 100 μM with a detection limit of 53 nM. The viability of this method for the monitoring of dopamine in spiked biological fluids was also established and it was successfully applied for the direct determination of dopamine in a blood serum and urine without preliminary treatment with satisfactory quantitative recovery 90-95% and 82-93%, respectively. The structural investigation using circular dichroism, fluorescence spectroscopy, and docking simulation indicated that, changes in the microenvironment of aromatic residues were significant, while minor conformational alterations of the protein were observed. It seems dopamine inhibits bioluminescence activity with specific binding to the residues involved in the light production.
关键词： therapeutic drug monitoring,dopamine,aequorin,photoprotein inhibition assay
更新于2025-09-23 15:21:01