Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay.
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.
Feltus
A
A
Department of Chemistry, University of Kentucky, Lexington 40506, USA.
Grosvenor
A L
AL
Conover
R C
RC
Anderson
K W
KW
Daunert
S
S
D000818:Animals; D001710:Biotin / Q000032:analysis*; D008163:Luminescent Measurements; D009865:Oocytes / Q000737:chemistry; D012617:Sea Urchins
D001710:Biotin