Mitochondriale Isolation von der Skelettmuskulatur
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Summary
Abstract
Protocol
Discussion
Disclosures
The authors have nothing to disclose.
Acknowledgements
Materials
| 95% CO<sub>2</sub> / 5% O<sub>2</sub> mix | Local Gas Supplier | ||
| Adenosine 5′-diphosphate sodium salt | Sigma-Aldrich | A2754 | |
| Blue Rizla Paper | Hansatech | 890101 | |
| Bradford protein assay | Bio-Rad | 500-0006 | |
| Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) | Sigma-Aldrich | C2920 | |
| Centrifuge 5804R | Eppendorf | ||
| Compressed nitrogen | Local Gas Supplier | ||
| D-mannitol | Sigma-Aldrich | M9647 | |
| Ethlyene-glycol-bis-tetraacetic acid (EGTA) | Sigma-Aldrich | E3889 | |
| Ethylenediaminetetraacetic acid (EDTA) | Bio-Rad | 161-0728 | |
| Free fatty acid bovine serum albumin | Sigma-Aldrich | A8806 | |
| Glutamic acid | Sigma-Aldrich | G5889 | |
| HEPES sodium salt | Sigma-Aldrich | H7006 | |
| Isotemp 3006D | Fisher Scientific | ||
| Magnesium chloride | Sigma-Aldrich | M8266 | |
| Male Sprague Dawley Rats | Harlan Laboratories | 300-500g | |
| Malic acid | Sigma-Aldrich | M9138 | |
| Minifuge | ISC Bioexpress | C1301P | |
| Oligomycin | Sigma-Aldrich | O4876 | |
| Oxygen electrode disc | Hansatech | S1 | |
| Oxygraph | Hansatech | ||
| Oxygraph Plus V1.01 Software | Hansatech | ||
| pH-meter | Mettler Toledo | 1225506149 | |
| Phosphate-buffered saline (PBS) | Sigma-Aldrich | P4417 | |
| Potassium chloride | Sigma-Aldrich | P3911 | |
| Potassium phosphate | Sigma-Aldrich | P8416 | |
| Potter-Elvehjem homogenizers | Fisher Scientific | 08-414-14A | |
| PTFE (0.0125mm × 25mm) membrane | Hansatech | S4 | |
| SKIL 3320 drill press | Hardware store | ||
| Sucrose | Sigma-Aldrich | S5016 |
References
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- Pallotti, F., Lenaz, G. Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. Methods Cell Biol. 80, 3-44 (2007).
- Pallotti, F., Lenaz, G. Isolation and subfractionation of mitochondria from animal cells and tissue culture lines. Methods Cell Biol. 65, 1-35 (2001).
- Gamboa, J. L., Andrade, F. H. Mitochondrial content and distribution changes specific to mouse diaphragm after chronic normobaric hypoxia. Am J Physiol Regul Integr Comp Physiol. , (2009).
- Patel, S. P., Gamboa, J. L., McMullen, C. A., Rabchevsky, A., Andrade, F. H. Lower respiratory capacity in extraocular muscle mitochondria: evidence for intrinsic differences in mitochondrial composition and function. Invest Ophthalmol Vis Sci. 50, 180-186 (2009).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248-254 (1976).
Tags
Mitochondrial IsolationSkeletal MuscleOrganellesMetabolic ReactionsATP SynthesisSignaling CascadesLiverBrainHeartSkeletal Muscle FunctionIsolation MethodBuffersReagentsMitochondrial Protein ConcentrationFunctional StudiesMitochondrial RespirationPolarographyClark-type ElectrodeOxygen Electrode CalibrationExperimental BufferGlutamateMalateAdenosine Diphosphate (ADP)Oligomycin
Cite This Article
Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. Mitochondrial Isolation from Skeletal Muscle. J. Vis. Exp. (49), e2452, doi:10.3791/2452 (2011).