2001
DOI: 10.1021/ac001258a
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Detection of Biotin in Individual Sea Urchin Oocytes Using a Bioluminescence Binding Assay

Abstract: 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 oocyte… Show more

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Cited by 6 publications
(3 citation statements)
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“…A proteolytic bond cleavage assay has also been developed using an aequorin fusion protein (). Since aequorin can be detected down to attomole levels, it has been employed in the determination of biotin in single cells and in picoliter-volume vials ( ). In addition, aequorin-labeled probes have been used as an alternative to radiolabels in nucleic acid hybridization assays ( , ).…”
Section: Introductionmentioning
confidence: 99%
“…A proteolytic bond cleavage assay has also been developed using an aequorin fusion protein (). Since aequorin can be detected down to attomole levels, it has been employed in the determination of biotin in single cells and in picoliter-volume vials ( ). In addition, aequorin-labeled probes have been used as an alternative to radiolabels in nucleic acid hybridization assays ( , ).…”
Section: Introductionmentioning
confidence: 99%
“…The oocytes are giant cells about 1.2 mm in diameter, a live test-tube for short-term mechanistic studies of gene expression, apoptosis, metabolism, and hormone signaling [13,14]. A number of applications in oocytes employs BLI, including measurements of calcium levels in X. laevis [15], of ATP in human oocytes [16], of biotin in sea urchin oocytes [17], and transgene expression in porcine ovarian follicles [18]. Oocytes were microinjected into the nucleus with a Luc-expressing vector pVaxLuc, grouped 8 to 10 per well of the cell culture plate in medium supplied with 5 mM solution of Cycluc, or 100 mM of D-Luc, as concentrations relevant for the experimental practice.…”
Section: Resultsmentioning
confidence: 99%
“…Nonacridinium salt-based reagents have been described in luminescence assays that rely on quenching or energy transfer. Specifically, assays for biotin have been developed utilizing the bioluminescent photoprotein aequorin. Bioluminescence resonance energy transfer (BRET) has recently been demonstrated to be useful for studies of protein−protein interactions. Similarly, chemiluminescence resonance energy transfer assays utilizing aminobutylethylisoluminol (ABEI) as the luminescent donor and fluorescein as the acceptor have been successfully developed for several biological ligands of interest. …”
mentioning
confidence: 99%