Mitochondrial aconitase activity and reactivation modified for Drosophila.
Method Article
Drosophila aconitase activity and reactivation
https://doi.org/10.1038/nprot.2007.169
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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.
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