Anaspec has introduced a series of luminescent and non-luminescent calcium indicators.
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 oxidised to coelenteramide, with a concomitant release of carbon dioxide and blue light.
The approximately third-power dependence of aequorin's bioluminescence on Ca2+ concentration allows the measurement of Ca2+ concentrations with a detection range from 0.1uM to >100uM.
Unlike fluorescent Ca2+ indicators, Ca2+ bound aequorin can be detected without illuminating the sample, thereby eliminating interference from autofluorescence and allowing simultaneous labelling with caged probes.
Anaspec offers coelenterazine and several synthetic coelenterazine analogues for reconstituting aequorin in cells that have been transfected with apoaequorin CDNA.
In addition to native coelenterazine, the company also offers 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 analogues 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 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.
