Mitochondrial aconitase activity and reactivation modified for Drosophila.
Method Article
Drosophila aconitase activity and reactivation
https://doi.org/10.1038/nprot.2007.169
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 20 Mar, 2007
You are reading this latest protocol version
aconitase
reactivation
iron-sulfur center
dithiothreitol
Mitochondrial aconitase activity and reactivation modified for Drosophila.
Sonicate mitochondria four times for 15 seconds in a Branson 450 sonicator in aconitase buffer: 50 mM Tris, 30 mM sodium citrate, 0.5 mM MnCl2, 0.2 mM NADP, pH 7.3.
Monitor the conversion of citrate into alpha-ketoglutarate at 340 nm at 25°C using the coupled reduction of NADP to NADPH by 2 units/ml of isocitrate dehydrogenase in 50 mM Tris, 1 mM cysteine, 1 mM sodium citrate, 0.5 mM MnCl2 at pH 7.4.
Reactivate aconitase by incubation with 2 mM dithiothreitol and 0.2 mM ferrous ammonium sulfate for 5 min then repeat the enzymatic activity assay.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 20 Mar, 2007
You are reading this latest protocol version
© Research Square 2022