Assay aequorin

Assay of aequorin. The assay of aequorin is simple. To a vial containing a small amount of aequorin sample (1-100 xl), 1 ml of 10 mM calcium acetate solution is injected, measuring the total amount of light emitted. The amount of the total light is proportional to the amount of aequorin in the sample. 

Minute amounts of coelenterazine can also be measured utilizing apoaequorin or apoobelin (Campbell and Herring, 1990 Thompson et ah, 1995). In this method, a sample containing coelenterazine is treated with an excess amount of apophotoprotein (apoaequorin or apoobelin) to convert it to a Ca2+-sensitive photoprotein (aequorin or obelin). The photoprotein formed is assayed by luminescing it with Ca2+ to determine the amount of coelenterazine originally existed. With this method, the luminescence reaction is fast and usually complete in a few seconds, in contrast to the slower luminescence reactions with luciferases that sometimes require a few minutes to complete. However, the formation of photoprotein from apoaequorin is slow and not necessarily quantitative, and the overall accuracy of the photoprotein method does not compare favorably with that of the luciferase method that directly measures coelenterazine. The author recommends using a luciferase if the enzyme is available. 

Blinks, J. R., et al. (1978). Practical aspects of the use of aequorin as a calcium indicator Assay, preparation, microinjection, and interpretation of signals. Method. Enzymol. 57 292-328. 

Stults, N. L., et al. (1992). Use of recombinant biotinylated aequorin in microtiter and membrane-based assays Purification of recombinant aequorin from Escherichia coli. Biochemistry 31 1433-1442. 

Acantbephyra, 162, 336 Acantboscina, 336 Acholoe, 335 Achromobacter, 35, 36 Acorn worms (enteropneusts), 315 Acylhomoserine lactone, 43 Advice to students, 375 Aequorea, 159, 161, 162, 334, 375 Aequorea aequorea, 92-94, 346 collection, 93, 94 distribution, 92 squeezate, 94 synonyms, 92 Aequorea GFP, 150-154 chromophore, 153 cloning, 154 crystallization, 130 fluorescence quantum yield, 152 isolation, 129 molecular weight, 152 spectral properties, 130, 152 Aequorea victoria, 92 Aequorin, 92-129, 159, 160,172,173, 175, 346, 349, 350, 364, 375 assay, 98... 

The jellyfish Aequorea contains a photoprotein, which emits light only when calcium ions are present.672 673 Since light emission can be measured with great sensitivity (modern photomultipliers can be used to count light quanta) the protein aequorin and related photoproteins674a are used as a sensitive indicator of calcium ion concentration.674 (In a similar way the firefly luciferin-luciferase system, which requires ATP for activation, is widely used in an assay for ATP.)... 

Dupriez, V.J. et al. 2002. Aequorin-based functional assays for G protein-coupled receptors, ion channels, and tyrosine kinase receptors. Recept. Chann. 8, 319-330. 

High throughput flash luminescence readers such as the FDS6000 and 7000, as well as the Lumax Flash HT, enable functional GPCR and calcium channel testing. A sensitive photon-counting CCD camera enables aequorin and luciferase activity to be measured by flash luminescence. Thus, these advances allow fluorescence-based assays to be replaced by luminescence-based assays,... 

Zatta PE, Nyame K, Cormier Ml, Mattox SA, Prieto PA, Smith DF, Cummings RD. A solid-phase assay for beta-1,4-gaIactosyI-transferase activity in human serum using recombinant aequorin. Anal. Biochem. 1991 194 185-191. 

Immunometric assays for TSH are available commercially as manual kit procedures or for use on automated systems. For practical reasons, nonisotopic methods dominate the market and have replaced radioactive tracer methods in most routine laboratories. The majority of immunometric TSH assays label the detection antibody with chemiluminescent labelled molecules other labels include peroxidase or alkaline phosphatase and sensitive photo-metric and fluorescenri molecules. Other assays are based on the use of fluorescent labels using europium chelates chemiluminescent compounds such as acri-dinium esters or ruthenium or bioluminescent molecules such as recombinant aequorin. ... 

Assays utilizing coelenterazine-charged aequorin as a probe for intracellular calcium level have been used to investigate a wide variety of receptors endothehn ETa, angiogensin ATn, tyrosine-releasing hormone (TRH), and neurokinin NKi [170] 5-hydroxytryptamine 2A [171], and human EPl prostanoid receptor [172]. 

RAPID AND SIMULTANEOUS BIOLUMINESCENT ASSAY OF AEQUORIN AND FIREFLY LUCIFERASE... 

We have developed a highly sensitive simultaneous bioluminescent assay of firefly luciferase and aequorin. Firefly luciferin-luciferase reaction is specific and sensitive for the determination of ATP, and this reaction has been widely used, e.g. for hygiene monitoring. Aequorin bounds specifically to Ca and then emits blue light, thus aequorin is useful to study intercellular We thought that these photoproteins... 

Simultaneous bioluminescent assay for aequorin and biotinylated luciferase... 

In the proposed assay, there was no interference in the measurement of the aequorin activity by biotinylated luciferase. The sensitivity of aequorin was 7.58xl0 0 mol/assay (blank + 3SD, Fig. 2, left), the calibration linearity of the assay ranged from 1.21 X lO mol/assay to 1.94 x lO mol/assay and the intra-assay coefficients of variation (CV) for 8 replicates with each standard point were from 1.0 to 3.2%. These results are similar to the results from the separate assay. 

Biotinylated luciferase and aequorin have been used as reported labelling protein for immunoassay. Thus, the proposed simultaneous assay of aequorin and biotinylated luciferase could be applied to immunoassay. We apply the proposed assay to tandem immunoassay using these two proteins as labels. 

Kozlova O, Zwinderman M, Christofi N. A new short term toxicity assay using Aspergillus awamori with recombinant aequorin gene. BMC Microbiol 2005 5 40-2. 

---