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Isolation of mitochondria from rat brain using Percoll density gradient centrifugation
Neil R Sims1, Michelle F Anderson
1Centre for Neuroscience and Department of Medical Biochemistry, School of Medicine, Flinders University, GPO Box 2100, Adelaide, South Australia 5001, Australia. Neil.Sims@flinders.edu.au
Nature Protocols
|July 5, 2008
Summary
Researchers developed new methods to isolate pure mitochondria from rat brains using Percoll density gradient centrifugation. These techniques yield well-coupled mitochondria with high respiratory activity, suitable for various research needs.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Mitochondria are crucial for cellular energy production.
- Isolating pure mitochondria from brain tissue is challenging due to contamination.
- Previous methods often result in compromised mitochondrial function or purity.
Purpose of the Study:
- To develop and present optimized protocols for isolating intact and functional mitochondria from rat brain tissue.
- To minimize contamination from synaptosomes and myelin during mitochondrial isolation.
- To offer variations of the isolation procedure suitable for different sample sizes and research requirements.
Main Methods:
- Combined differential centrifugation and discontinuous Percoll density gradient centrifugation.
- Utilized Percoll density gradients to separate mitochondria from other cellular components.
- Included a variation employing digitonin to disrupt synaptosomes prior to isolation for small samples.
Main Results:
- Achieved high purity mitochondrial preparations with minimal synaptosome and myelin contamination.
- Isolated mitochondria demonstrated excellent coupling and high respiratory activity.
- Developed three protocol variations with differing yields, preparation times, and sample size suitability.
Conclusions:
- The presented procedures effectively isolate functional mitochondria from rat brains.
- Percoll density gradient centrifugation is key to achieving high-purity mitochondrial isolates.
- The optimized protocols provide reliable methods for neuroscience and cell biology research.
