YILIMART - Mito MidiMACS Starting Kit, mouse tissue

The Mitochondria Isolation Kit, mouse tissue facilitates the isolation of functional and viable mitochondria from mouse tissue. The isolation protocol is based on the renowned MACS Technology, which enables fast isolation of high purity and high yield mitochondria.

Detailed procedure

Tissues can be homogenized with the gentleMACS or gentleMACS Octo Dissociator in combination with the Mitochondria Extraction Kit – Tissue. After one-step homogenization of the tissue and lysis of the cell suspension, mitochondria are magnetically labeled with Anti-TOM22 MicroBeads, mouse, which bind to the translocase of the outer mitochondrial membrane 22 protein (TOM22). The sample is loaded onto an LS Column placed in a MidiMACS or QuadroMACS Separator. After washing, only the magnetically labeled mitochondria are retained on the column. The column is removed from the separator and functional mouse mitochondria are eluted from the column.

Downstream applications

After isolation mitochondria can be further subjected to Western blot analysis

^1,6

or downstream functional assays, including measurement of O2, membrane potential, and ATP

^4-5,7

, or respiratory control

⁴

. Growing evidence also suggests that isolated mitochondria are well suited for mitochondrial RNA expression profiling studies

^2,3

.

Figure 1
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Figure 1

Enrichment of functional mitochondria. Mitochondria were prepared from various mouse tissues using the Mitochondria Isolation Kit, mouse tissue. The mitochondria preparations from muscle, liver, and brain, as well as the flow-through fractions were analyzed by a coupled enzymatic citrate synthase assay.

The data indicate that citrate synthase activity, normalized to the activity per mg protein assayed in the flow-through fraction, is highly enriched in the eluate.

Enrichment of functional mitochondria. Mitochondria were prepared from various mouse tissues using the Mitochondria Isolation Kit, mouse tissue. The mitochondria preparations from muscle, liver, and brain, as well as the flow-through fractions were analyzed by a coupled enzymatic citrate synthase assay.

The data indicate that citrate synthase activity, normalized to the activity per mg protein assayed in the flow-through fraction, is highly enriched in the eluate.
Figure 2
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Figure 2

Integrated tissue dissociation followed by mitochondria isolation.

Integrated tissue dissociation followed by mitochondria isolation.
Figure 3
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Figure 3

Isolation of high-purity mitochondria from mouse liver. After isolation of mitochondria from mouse liver using the Mitochondria Isolation Kit, mouse tissue, equal amounts of mitochondrial protein lysates were analyzed by Western blotting against TOM20. PEX1, a cytoplasmic protein anchored to peroxisomal membranes, served as a control.

The results show an effective enrichment of mitochondria and no contamination of the mitochondria fraction by peroxisomes.

Isolation of high-purity mitochondria from mouse liver. After isolation of mitochondria from mouse liver using the Mitochondria Isolation Kit, mouse tissue, equal amounts of mitochondrial protein lysates were analyzed by Western blotting against TOM20. PEX1, a cytoplasmic protein anchored to peroxisomal membranes, served as a control.

The results show an effective enrichment of mitochondria and no contamination of the mitochondria fraction by peroxisomes.

Selected references
1. Hornig-Do, H.T. et al. (2009) Isolation of functional pure mitochondria by superparamagnetic microbeads. Anal. Biochem. 389(1): 1-5
2. Guo, T. et al. (2010) Quantitative proteomics discloses MET expression in mitochondria as a direct target of MET kinase inhibitor in cancer cells. Mol. Cell. Proteomics 9(12): 2629-2641
3. Minet, A.D. and Gaster, M. (2010) ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects. Biochem. Biophys. Res. Commun. 402(1): 70-74
4. Bandiera, S. et al. (2011) Nuclear outsourcing of RNA interference components to human mitochondria. PLoS One 6(6): e20746
5. Barrey, E. et al. (2011) Pre-microRNA and mature microRNA in human mitochondria. PLoS One 6(5): e20220
6. Eriksen, M. B. et al. (2011) Intact primary mitochondrial function in myotubes established from women with PCOS. J. Clin. Endocrinol. Metab. 96(8): E1298-1302
7. Minet, A.D. and Gaster, M. (2011) The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control. Biochem. Biophys. Res. Commun. 409(4): 591-595
8. Sacchi, S. et al. (2011) Evidence for the interaction of d-amino acid oxidase with pLG72 in a glial cell line. Mol. Cell. Neurosci. 48(1): 20-28
9. Hornig-Do, H.T. et al. (2012) Nonsense mutations in the COX1 subunit impair the stability of respiratory chain complexes rather than their assembly. EMBO J. 31(5): 1293-1307
10. Mukhopadhyay, P. et al. (2012) Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: therapeutic potential of mitochondrially-targeted antioxidants. Free Radic. Biol. Med. 53(5): 1123-1138
11. Papkovskaia, T.D. et al. (2012) G2019S leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization. Hum. Mol. Genet. 21(19): 4201-4213
12. Schiller, M. et al. (2012) Induction of type I IFN is a physiological immune reaction to apoptotic cell-derived membrane microparticles. J. Immunol. 189(4): 1747-1756
Brochures and posters

Mitochondria isolation
Customer reports and application notes

Synaptic mitochondria isolation

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Mito MidiMACS Starting Kit, mouse tissue

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Detailed procedure

Downstream applications

Figure 1

Figure 1

Figure 2

Figure 2

Figure 3

Figure 3

Selected references

Brochures and posters

Customer reports and application notes

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