San Jose, CA – November 12, 2008
The aequorin complex comprises a 22 KD apoaequorin protein (APO), molecular oxygen and the luminophore coelenterazine. When three Ca2+ ions bind to this complex, coelenterazine is oxidized to coelenteramide, with a concomitant release of carbon dioxide and blue light (See figure below). The approximately third-power dependence of aequorin’s bioluminescence on Ca2+ concentration allows the measurement of Ca2+ concentrations with a broad detection range from ~0.1 µM to >100 µM. Unlike fluorescent Ca2+ indicators, Ca2+-bound aequorin can be detected without illuminating the sample, thereby eliminating interference from autofluorescence and allowing simultaneous labeling with caged probes.
AnaSpec offers coelenterazine and several synthetic coelenterazine analogs for reconstituting aequorin in cells that have been transfected with apoaequorin cDNA. In addition to native coelenterazine, we also offer a few derivatives of coelenterazine that confer different Ca2+ affinities and spectral properties on the aequorin complex. Recombinant apoaequorin reconstituted with coelenterazine hcp is reported to have the best luminescence overall, with both a high quantum yield and a fast response time. However, intracellular reconstitution of aequorin from coelenterazine analogs can be relatively slow. Aequorins containing the cp, f or h form of coelenterazine exhibit 10–20 times stronger luminescence than that of apoaequorin reconstituted with native coelenterazine. Coelenterazine cp has been used in HTS screening assay for GPCRs.
Intracellular calibration of Ca2+ indicators may be achieved either by manipulating Ca2+ levels inside cells using an ionophore or by releasing the indicator into the surrounding medium of known Ca2+ concentration via detergent lysis of cells. Along with fluorescent and luminescent Ca2+ detection reagents, AnaSpec also offers several non-luminescent compounds for measuring and manipulating intracellular and extracellular Ca2+. Amongst these, 5,5’-dimethyl BAPTA is the most powerful Ca2+ chelator, which is also available in cell-permeable AM ester form. 5,5’-difluoro BAPTA and its AM ester have been employed for optical imaging studies but are most widely used for NMR analysis of Ca2+ in live cells and tissues.
AnaSpec is pleased to showcase a wide range of reagents for luminescent Ca2+ detection and non-luminescent reagents for Ca2+ detection.
About AnaSpec
AnaSpec is a leading provider of integrated proteomics solutions to the world’s largest biotech, pharmaceutical, and academic research institutions. With a vision for innovation through synergy, AnaSpec focuses on three core technologies: peptides, detection reagents, and combinatorial chemistry.
For more information visit
www.anaspec.com###
