Biologists are constantly seeking more sensitive assays to detect the presence of organisms or molecules, including DNA, RNA, and proteins. Fluorescent labels are not always sensitive enough for a particular detection need.
In those cases, chemiluminescence and bioluminescence detection can be the best choice. Although radioactive labels generally offer the sensitivity required, their safety and disposal issues make them less than ideal choices. Luminescence systems rely on a reporter enzyme which signals the presence of the target and produces the luminescence, and a substrate. The substrate is processed by the reporter enzyme, resulting in a characteristic flash or glow.
Luminescence systems have been adapted to many of the protocols that use fluorescent and radiolabeled tags, including DNA and RNA probes, DNA/RNA sequencing, and chromosome imaging. The sensitivity and flexibility of luminescence systems have spawned other uses. For example, luciferase modifies adenosine triphosphate (ATP) and its substrate luciferin, which produces light, and Aequorin, a photoprotein isolated from jellyfish and other marine organisms, produces light upon calcium binding.
Aequorin is comprised of the apoprotein apoaequorin, and coelenterazine, a luciferin. These two components reconstitute spontaneously, forming the functional protein. The protein has three calcium binding sites. When calcium occupies these sites, the protein undergoes a conformational change which converts coelenterazine into excited coelenteramide. As coelenteramide relaxes to the ground state, blue light is emitted, which can be detected via a luminometer or luminescence microplate reader. As a result, Aequorin has become a useful tool for the measurement of intracellular calcium levels in biological systems.
To learn more about bioanalytical systems, software, equipment and instruments from Molecular Devices that detect luminescence, visit the links below.
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