Real-Time Fluorescence Detection of Calcium Efflux During Vacuolar Membrane Fusion.
Methods Mol Biol
; 1860: 323-331, 2019.
Article
em En
| MEDLINE
| ID: mdl-30317515
ABSTRACT
During in vitro homotypic yeast vacuole fusion Ca2+ is transported into and out of the organelle lumen. In vitro, Ca2+ is taken up from the medium by vacuoles upon the addition of ATP. During the docking stage of vacuole fusion Ca2+ is effluxed from the lumen upon the formation of trans-SNARE complexes between vesicles. Here we describe a real-time fluorescence-based assay to monitor the transport of this cation using purified organelles. Extraluminal Ca2+ is detected when the cation binds the low-affinity fluorescent dye Fluo-4 dextran. This allows for the use of a 96-well microtiter plate to be read in a fluorescence plate reader. Thus, in addition to a curve of calibrated Ca2+ standards, up to 91 experimental conditions can be monitored in a single microplate using this method.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Bioensaio
/
Cálcio
/
Proteínas de Saccharomyces cerevisiae
/
Proteínas SNARE
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article