Drosophila aconitase activity and reactivation
Mitochondrial aconitase activity and reactivation modified for Drosophila.
1) 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.
2) 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.
3) Reactivate aconitase by incubation with 2 mM dithiothreitol and 0.2 mM ferrous ammonium sulfate for 5 min then repeat the enzymatic activity assay.
very useful!
It would be useful to mention the incubation temperature, e.g., Bubber et al (in Neurochemistry International 2004, 45: 1021 – 28 and presumably this one as well - Annals of Neurology 2005, 57: 695 – 703) reactivate [mouse] aconitase at 37°C. It would also help if other conditions were described, such as if anaerobic conditions were maintained during reactivation (e.g. use of glove bags or replacing air with nitrogen/argon in tubes). I assume this procedure was conducted at room temperature in aerobic conditions, which would require operator to carry out assay immediately to prevent loss of activity in a time dependent manner due to aerial oxidation.
very useful!
It would be useful to mention the incubation temperature, e.g., Bubber et al (in Neurochemistry International 2004, 45: 1021 – 28 and presumably this one as well - Annals of Neurology 2005, 57: 695 – 703) reactivate [mouse] aconitase at 37°C. It would also help if other conditions were described, such as if anaerobic conditions were maintained during reactivation (e.g. use of glove bags or replacing air with nitrogen/argon in tubes). I assume this procedure was conducted at room temperature in aerobic conditions, which would require operator to carry out assay immediately to prevent loss of activity in a time dependent manner due to aerial oxidation.
Posted 20 Mar, 2007
Drosophila aconitase activity and reactivation
Posted 20 Mar, 2007
Mitochondrial aconitase activity and reactivation modified for Drosophila.
1) 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.
2) 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.
3) Reactivate aconitase by incubation with 2 mM dithiothreitol and 0.2 mM ferrous ammonium sulfate for 5 min then repeat the enzymatic activity assay.
very useful!
It would be useful to mention the incubation temperature, e.g., Bubber et al (in Neurochemistry International 2004, 45: 1021 – 28 and presumably this one as well - Annals of Neurology 2005, 57: 695 – 703) reactivate [mouse] aconitase at 37°C. It would also help if other conditions were described, such as if anaerobic conditions were maintained during reactivation (e.g. use of glove bags or replacing air with nitrogen/argon in tubes). I assume this procedure was conducted at room temperature in aerobic conditions, which would require operator to carry out assay immediately to prevent loss of activity in a time dependent manner due to aerial oxidation.
very useful!
It would be useful to mention the incubation temperature, e.g., Bubber et al (in Neurochemistry International 2004, 45: 1021 – 28 and presumably this one as well - Annals of Neurology 2005, 57: 695 – 703) reactivate [mouse] aconitase at 37°C. It would also help if other conditions were described, such as if anaerobic conditions were maintained during reactivation (e.g. use of glove bags or replacing air with nitrogen/argon in tubes). I assume this procedure was conducted at room temperature in aerobic conditions, which would require operator to carry out assay immediately to prevent loss of activity in a time dependent manner due to aerial oxidation.
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