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Protocol to characterize mitochondrial supercomplexes from mouse tissues by combining BN-PAGE and MS-based
Roger Moreno-Justicia1, Alba Gonzalez-Franquesa1, Ben Stocks1
1Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen 2200, Denmark.
STAR Protocols
|February 7, 2022
Summary
This protocol isolates mitochondria and uses blue native gel electrophoresis to analyze mitochondrial supercomplexes. It enables efficient measurement of protein abundance and distribution within these essential cellular structures.
Area of Science:
- Mitochondrial biology
- Proteomics
- Biochemistry
Background:
- Mitochondrial respiratory complexes assemble into supercomplexes, impacting cellular energy production.
- Understanding the composition and dynamics of these supercomplexes is crucial for mitochondrial function.
- Existing methods for analyzing supercomplexes can be time-consuming.
Purpose of the Study:
- To provide a detailed protocol for the isolation and analysis of mitochondrial supercomplexes.
- To enable efficient measurement of protein abundance and distribution within known supercomplexes.
- To facilitate MS-based proteomics of separated supercomplexes.
Main Methods:
- Mitochondrial isolation from mouse tissues.
- Blue native gel electrophoresis (BN-PAGE) for supercomplex separation.
- In-gel protein digestion and peptide desalting for mass spectrometry (MS).
Main Results:
- Successful separation of pre-identified mitochondrial supercomplexes into distinct gel bands.
- A time-efficient method for quantifying protein composition within supercomplexes.
- Enables detailed proteomic analysis of individual supercomplexes.
Conclusions:
- The described protocol offers an efficient approach to study mitochondrial supercomplexes.
- This method aids in understanding the functional implications of supercomplex assembly.
- Facilitates advancements in mitochondrial research through detailed proteomic analysis.