This protocol describes the use of the jellyfish Aequorea victoria aequorin protein to measure Ca2+ levels in living yeast cells. All yeast strains to be analyzed must express the A. victoria apoprotein of the aequorin calcium biosensor, to be reconstituted into fully active aequorin by association with its cofactor, coelenterazine, which cannot be synthesized by yeast itself. The simplest way to achieve reconstitution is to transform yeast cells with a vector driving apoaequorin expression, and then supply commercially available coelenterazine cofactor in the medium. Coelenterazine is a hydrophobic molecule and is able to permeate yeast cells.
Tisi, R., Martegani, E., Brandão, R. (2015). Monitoring yeast intracellular ca2+ levels using an in vivo bioluminescence assay. COLD SPRING HARBOR PROTOCOLS, 2015(2), 210-213 [10.1101/pdb.prot076851].
Monitoring yeast intracellular ca2+ levels using an in vivo bioluminescence assay
TISI, RENATA ANITA
Primo
;MARTEGANI, ENZOSecondo
;
2015
Abstract
This protocol describes the use of the jellyfish Aequorea victoria aequorin protein to measure Ca2+ levels in living yeast cells. All yeast strains to be analyzed must express the A. victoria apoprotein of the aequorin calcium biosensor, to be reconstituted into fully active aequorin by association with its cofactor, coelenterazine, which cannot be synthesized by yeast itself. The simplest way to achieve reconstitution is to transform yeast cells with a vector driving apoaequorin expression, and then supply commercially available coelenterazine cofactor in the medium. Coelenterazine is a hydrophobic molecule and is able to permeate yeast cells.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.