Mitochondria Isolation Kit

Western Blot: Mitochondria Isolation Kit [NBP2-29448] - Mouse liver cells were fractionated using 20 ug of cytosolic (lane 1) and mitochondrial (lane 2) extracts, probed with anti-Cytochrome C antibody, and anti-Actin antibody (loading control). Isolation kit efficiently separated the two fractions: No Cytochrome C (specific to mitochondria) was detected in the cytosolic extract.

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Mitochondria Isolation Kit Summary

• Description: This kit can be used to isolate cytosolic and mitochondrial fractions from the same cell or tissue preparations. The procedure is based on the principle of differential centrifugation. The high density nuclei are first removed by low-speed centrifugation on a sucrose cushion. The supernatant containing the mitochondria is subjected to high speed centrifugation to retrieve the mitochondria.
  
  This kit contains reagents required for the isolation of mitochondria from 10 grams of mammalian tissues (liver, muscle, etc.) or 100 reactions for cells grown in tissue culture plates. The mitochondrial and cytosolic fractions can be used for studying apoptotic and signal transduction pathways to detect translocation of any factors of interest within the two fractions by western blotting, ELISA, or other assay.
• Kit Type: Isolation Kit

Applications/Dilutions

• Dilutions:
  • Western Blot

Packaging, Storage & Formulations

• Storage: Storage of components varies. See protocol for specific instructions.

Alternate Names for Mitochondria Isolation Kit

• Mitochondria Extraction Kit

Background

NB600-556 recognizes a 60kDa non-glycosylated protein component of mitochondria found in human cells. It can be used to stain the mitochondria in cell or tissue preparations and can be used as a marker of the mitochondria in subcellular fractions. This monoclonal antibody is part of a new panel of reagents which recognizes subcellular organelles or compartments of human cells. These markers may be useful in identification of these organelles in cells, tissues, and biochemical preparations. MAb-2 produces a spaghetti-like staining pattern in the cytoplasm of normal and malignant human cells and may be used to stain the mitochondria in paraformaldehyde fixed frozen and formalin-fixed, paraffin-embedded tissues.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.

Publications for Mitochondria Kit (NBP2-29448)(22)

Publications using NBP2-29448

• Hwang DJ, Kwon KC, Choi DH Et al. Comparison of intrinsic exercise capacity and response to acute exercise in ICR (Institute of Cancer Research) mice derived from three different lineages Laboratory animal research [PMID: 34348800]
• Hornstein T. Molecular elucidation of apoptosis resistance in neutrophils from patients with severe trauma. Thesis (WB, Human)
• Dahal S, Dubey S, Raghavan SC. Homologous recombination-mediated repair of DNA double-strand breaks operates in mammalian mitochondria Cell. Mol. Life Sci. 2017-11-07 [PMID: 29116362] (Mouse)
• Chakraborty S, Ghosh S, Banerjee B et al. Mephebrindole, a synthetic indole analog coordinates the crosstalk between p38MAPK and eIF2a/ATF4/CHOP signalling pathways for induction of apoptosis in human breast carcinoma cells Apoptosis 2016-07-08 [PMID: 27392939] (Mouse)
• Tadi SK, Sebastian R, Dahal S et al. Microhomology Mediated End Joining is the Principal Mediator of Double-strand Break Repair During Mitochondrial DNA Lesions. Mol. Biol. Cell. 2015-11-25 [PMID: 26609070]
  
  Details:
  Mitochondrial Extraction Kit was used for the preparation of mitochondrial protein extracts from brain, testes, spleen, and kidney of rats and HeLa cells.
• Hornstein T, Lehmann S, Philipp D et al. Staurosporine resistance in inflammatory neutrophils is associated with the inhibition of caspase- and proteasome-mediated Mcl-1 degradation. J. Leukoc. Biol. 2015-08-26 [PMID: 26310832]
• Chong A, Vandana J, Li G Et Al. Checkpoint Kinase 2 Controls Insulin Secretion and Glucose Homeostasis SSRN Electronic Journal 2021-07-23
• Jeong JH, Koo JH, Yook JS, et al. Neuroprotective Benefits of Exercise and MitoQ on Memory Function, Mitochondrial Dynamics, Oxidative Stress, and Neuroinflammation in D-Galactose-Induced Aging Rats Brain sciences 2021-01-27 [PMID: 33514020]
• Dietl A, Winkel I, Pietrzyk G, et al. Skeletal muscle alterations in tachycardia-induced heart failure are linked to deficient natriuretic peptide signalling and are attenuated by RAS-/NEP-inhibition PLoS ONE 2019-12-04 [PMID: 31800630]
• Koo JH, Kang EB Effects of treadmill exercise on the regulatory mechanisms of mitochondrial dynamics and oxidative stress in the brains of high-fat diet fed rats J Exerc Nutrition Biochem 2019-03-31 [PMID: 31010272]
• Shah SZA, Zhao D, Taglialatela G et al. Combinatory FK506 and Minocycline Treatment Alleviates Prion-Induced Neurodegenerative Events via Caspase-Mediated MAPK-NRF2 Pathway Int J Mol Sci 2019-03-06 [PMID: 30845718] (WB)
• Kang L, Dai C, Lustig ME et al. Heterozygous SOD2 Deletion Impairs Glucose-Stimulated Insulin Secretion, but not Insulin Action in High Fat-Fed Mice. Diabetes. 2014-06-19 [PMID: 24947366] (WB, Mouse)
  
  Details:
  Fig 1B: Mitochondria isolated from gastrocnemius were used for WB
• Somasagara Ranganatha R, Hegde Mahesh, Chiruvella Kishore K et al. Extracts of strawberry fruits induce intrinsic pathway of apoptosis in breast cancer cells and inhibits tumor progression in mice. PLoS One. 2012-01-01 [PMID: 23071702] (WB)
• Hegde Mahesh, Karki Subhas S, Thomas Elizabeth et al. Novel levamisole derivative induces extrinsic pathway of apoptosis in cancer cells and inhibits tumor progression in mice. PLoS One. 2012-01-01 [PMID: 22970136] (WB)
• Csak T, Dolganiuc A, Kodys K et al. Mitochondrial antiviral signaling protein defect links impaired antiviral response and liver injury in steatohepatitis in mice. Hepatology. 2011-06-01 [PMID: 21425308] (WB)
  
  Details:
  Cytochrome c (IMG-101A), Caspase-8 (IMG-5703), Mitochondrial extraction kit (10082k). WB: Mitochondrial and cytoplasmic extract from C57B16 MCD (methionine-choline deficient) diet fed and non MCD diet fed mice, Fig 5.
• Fu HY, Okada K, Liao Y et al. Ablation of C/EBP homologous protein attenuates endoplasmic reticulum-mediated apoptosis and cardiac dysfunction induced by pressure overload. Circulation. 2010-07-27 [PMID: 20625112] (Mouse)
  
  Details:
  Mitochondrial extraction kit (10082k). Mitochondrial fractions from mouse heart were subject to western blot using Bcl2 and Bax antibodies, Fig 4A.
• Cornille E, Abou-Hamdan M, Khrestchatisky M et al. Enhancement of L-3-hydroxybutyryl-CoA dehydrogenase activity and circulating ketone body levels by pantethine. Relevance to dopaminergic injury. BMC Neurosci. 2010-04-23 [PMID: 20416081]
  
  Details:
  Mitochondrial extraction kit (10082k). Mitochondrial extract was taken from brain and liver tissues.
• Lee S, Van Remmen H, Csete M. Sod2 overexpression preserves myoblast mitochondrial mass and function, but not muscle mass with aging. Aging Cell. 2009-06-01 [PMID: 19627269]
  
  Details:
  Mitochondrial extraction kit (10082k). Mitochondrial fractions were obtained from human myoblasts.
• Mancini F, Di Conza G, Pellegrino M et al. MDM4 (MDMX) localizes at the mitochondria and facilitates the p53-mediated intrinsic-apoptotic pathway. EMBO J. 2009-07-08 [PMID: 19521340] (WB)
  
  Details:
  Mitochondrial extraction kit (10082k). WB: Mitochondrial fractions from mouse embryonic fibroblasts (MEF) were subject to western blot, Fig 4.
• Leem YH, Lim HJ, Shim SB et al. Repression of tau hyperphosphorylation by chronic endurance exercise in aged transgenic mouse model of tauopathies. J Neurosci Res. 2009-08-15 [PMID: 19360903] (WB, Mouse)
  
  Details:
  Mitochondrial extraction kit (10082k). Mitochondrial fractions from NSE/htau23 mouse brain was subjected to western blot, Fig 1B.
• Diao J, Allister EM, Koshkin V et al. UCP2 is highly expressed in pancreatic alpha-cells and influences secretion and survival. Proc Natl Acad Sci U S A. 2008-08-19 [PMID: 18701716] (WB)
  
  Details:
  Isolation of mitochondrial and cytosolic fractions from mouse pancreatic cell lines, alpha-TC6 and MIN6 using 10082k: Fig 2, mitochondrial and cytosolic fractions were used for western blot.
• Ayala JE, Bracy DP, Julien BM et al. Chronic treatment with sildenafil improves energy balance and insulin action in high fat-fed conscious mice. Diabetes. 2007-04-01 [PMID: 17229936] (WB, Mouse)
  
  Details:
  Isolation of mitochondria from intacapsular brown fat (mice) using 10082k, Fig 3: Mitochondrial extracts were used for western blot.

FAQs for Mitochondria Kit (NBP2-29448). (Showing 1 - 1 of 1 FAQs).

1. We are looking to isolate mtDNA that is free from nuclear DNA and nuclear DNA that is free from mtDNA. Do you know if these kits (NBP2-29447, NBP2-29448) will be able to do this? We are wanting the isolated DNA to use as a positive control for a copy number experiment.
   • You would need to use both kits in order to seperate mtDNA and nDNA. I was worried about the lysis buffer contained in both kits would force the DNA to precipitate, this can occur if the buffer is very basic or alcohol based. I also double checked to make sure all the buffers contained within the kit do not have any DNAses or RNAses present. Follow the protocol for both kits until you use the mitochondrial/nuclear lysis buffer and then proceed to collect the individual supernatants. I would proceed to use our Quik ChIP Kit. The way this kit works is by precipitating the DNA and then running it through a spin column included into the kit. Then the DNA is eluded of the column and the resulting product should be purified mDNA or nDNA depending on which supernatant you used. I reccomend on using a lot of starting material for your nuclear extract as the yield tends to be low, which is typical. I also reccomend to use 18 mega ohm DI water when starting your nuclear/mitochondrial extractions. The nuclear and mitochondrial extraction kits were designed for the purpose of extracting proteins and have not been tested for this particular application.