Nature Protocols: Drosophila Aconitase activity and reactivation

This Protocol is listed in the following Categories:
Biochemistry and protein analysis, Cell and developmental biology, Isolation, purification and separation, Model organisms

Author(s): James Jiayuan Tong and Douglas C Wallace
Lab/Group: Wallace Lab (Irvine)
DOI: 10.1038/nprot.2007.169

Drosophila Aconitase activity and reactivation

Tong James Jiayuan, tongja@uci.edu, Biophysics and Physiology, UC Irvine

Wallace Douglas C, dwallace@uci.edu, MAMMAG, UC Irvine

Lab/Group: Wallace Lab (Irvine)

Related Journal & Article Information

Journal: Nature Genetics

Article Title: Life extension through neurofibromin mitochondrial regulation and antioxidant therapy for neurofibromatosis-1 in Drosophila melanogaster

Introduction

Mitochondrial aconitase activity and reactivation modified for Drosophila

Materials

Reagents

Equipment

Time Taken

Procedure

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.

Drosophila Aconitase activity and reactivation

Related Journal & Article Information

Introduction

Materials

Reagents

Equipment

Time Taken

Procedure

Troubleshooting

Critical Steps

Anticipated Results

References

Acknowledgements

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