Calibration of Micropipets Using the Bioluminescent Protein Aequorin

Grosvenor, Anne L.; Crofcheck, Czarena; Anderson, Kimberly W.; Daunert, Sylvia

doi:10.1021/ac970123a

Cited by 11 publications

(8 citation statements)

References 12 publications

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“…It has also been used as a highly sensitive label in binding assays (8)(9)(10)(11)(12) and in the detection of proteolytic bond cleavage (13). Aequorin can be detected in the femtomole to attomole range, which has allowed its employment as an alternative label to fluorescent probes in the calibration of micropipets (14). This photoprotein offers several advantages such as no photobleaching and the production of signal with virtually no background.…”

Section: Introductionmentioning

confidence: 99%

C-Terminal and N-Terminal Fusions of Aequorin with Small Peptides in Immunoassay Development

2001

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Aequorin fusion proteins have been used extensively in intracellular Ca2+ measurements and in the development of binding assays. Gene fusions to aequorin for production of fusion proteins have been so far limited to its N-terminus, as previous studies have indicated that aequorin loses its activity upon modification of its C-terminus. To further investigate this, two model peptides, an octapeptide (DTLDDDDL), and leu-enkephalin (TGGFL), an opioid peptide, were fused to the C-terminus of a cysteine-free mutant of aequorin through genetic engineering. The octapeptide was also fused to the N-terminus of the aequorin-leu-enkephalin fusion protein, which enables its affinity purification. Contrary to reports of earlier studies, we found that aequorin retains its bioluminescence activity after modification of the C-terminus. The half-life of light emission and the calibration curves obtained with the fusion proteins were comparable to those of the cysteine-free mutant of aequorin. Dose-response curves for the octapeptide were generated using two aequorin-octapeptide fusion proteins with the octapeptide fused to the N-terminus in one case, and to the C-terminus in the other. Similar detection limits for the octapeptide were obtained using both fusion proteins. The C-terminal fusion system has advantages in cases where antibodies recognize only the C-terminus of the peptide, as well as in cases where the functionality of the peptide lies in its C-terminus. The purification is also simplified as the affinity tag can be engineered at one terminus and the peptide of interest at the other.

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Section: Introductionmentioning

confidence: 99%

C-Terminal and N-Terminal Fusions of Aequorin with Small Peptides in Immunoassay Development

2001

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“…One important factor in the performance of an assay in such small volumes is the amount of error that is introduced during the delivery of the assay components. Previous results have indicated that the single largest source of error in these assays lies in microinjection of the reagents. − For this reason, we developed a protocol minimizing the number of injections necessary to perform the assay. This involves first injecting the AEQ−biotin (and free biotin, if needed) from one micropipet, followed by injection of the avidin mixed with Ca 2+ from a second micropipet.…”

Section: Resultsmentioning

confidence: 99%

“…BF 100-78-15) having an outside diameter of 1.0 mm and an inside diameter of 0.78 mm. Volume determination experiments are described in detail in ref 36. Briefly, injection volume was determined by measuring the diameter of the bubble created upon injection of the aqueous reagent into microscope immersion oil.…”

Section: Experimental Protocolmentioning

confidence: 99%

Detection of Biotin in Individual Sea Urchin Oocytes Using a Bioluminescence Binding Assay

et al. 2001

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The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

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“…So, in chemiluminescence the problems such as source instability and backgrounds noise are resolved. 4 Furthermore, chemiluminescence labeling which is commonly used in immunoassays has advantages such as fast light emission, 5 high sensitivity and safety, a controllable emission rate and low cost. In addition, conjugation makes the chemiluminant reagents more stable and also luminometers based on photomultiplier tubes are available.…”

Section: Introductionmentioning

confidence: 99%

A gold nanoparticle-based immunosensor for the chemiluminescence detection of the hepatitis B surface antigen

et al. 2014

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Calibration of Micropipets Using the Bioluminescent Protein Aequorin

Cited by 11 publications

References 12 publications

C-Terminal and N-Terminal Fusions of Aequorin with Small Peptides in Immunoassay Development

C-Terminal and N-Terminal Fusions of Aequorin with Small Peptides in Immunoassay Development

Detection of Biotin in Individual Sea Urchin Oocytes Using a Bioluminescence Binding Assay

A gold nanoparticle-based immunosensor for the chemiluminescence detection of the hepatitis B surface antigen

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