Mitochondria/Cytosol Fractionation Kit Sufficient for analysis of 50 samples Cat. No. MIT1000 FOR RESEARCH USE ONLY Not for use in diagnostic procedures. USA & Canada Phone: +1(800) 437-7500 Fax: +1 (951) 676-9209 www.millipore.com
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Introduction Mitochondria, sometimes described as the power plants of the cell, are sites where most of the energy production in eukaryotic cells takes place. The synthesis of most of the adenosine triphosphate (ATP) occurs in these double membraned organelles that are found in living cells. ATP production by the mitochondria is done by the process of respiration, which uses oxygen to generate energy. This is a very efficient process for using food energy to make ATP. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Mitochondria also regulate crucial apoptosis signaling pathways. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a few mitochondria, whereas others can contain several thousand. Millipore s Mitochondria/Cytosol Fractionation Kit provides reagents for quick and efficient isolation of intact mitochondria from cultured cells. This kit allows mitochondrial isolation by using a convenient table top microcentrifuge, and can be used to separate an enriched mitochondrial fraction (heavy membrane fraction) from cytosolic fraction (light membrane fraction). Such separation is useful for studying apoptosis and signaling pathways between the two fractions, by Western blotting or ELISA. Kit Components 1. Isotonic Mitochondrial Buffer (CS204255): 50 ml 2. Mitochondrial Lysis Buffer (CS204257): 5 ml 3. Protease inhibitor cocktail (CS204253): 1 ml 4. Anti-Bcl2 (05-729-25UG): 25 µg 5. Anti-GAPDH (CS204254): 25 µg Storage All components should be store at -20 C for up to o ne year from date of receipt.
Assay Instructions 1. Culture cells in 10 cm tissue culture dishes until confluent (~2x 10 7 cells per plate). 2. Add protease inhibitor cocktail to Isotonic Mitochondrial Buffer at 1:100 dilution. 3. Wash the cells twice with ice-cold PBS. Remove PBS and add 1mL of Isotonic Mitochondrial Buffer (containing protease inhibitors). 4. Use a cell scraper to detach the cells from the culture dish. 5. Homogenize the cells with 40 strokes in a Dounce homogenizer on ice (the use of a PTEF pestle bottom tissue grinder is recommended). 6. Centrifuge the lysate at 600 x g for 10 minutes at 4 C to pellet the nuclei and unbroken cells. 7. Transfer the supernatant to a fresh 1.5 ml Eppendorf tube, and centrifuge at 10,000 x g (~15000rpm) for 30 minutes at 4 C. 8. Collect the supernatant (cytosol and microsome fraction - light membrane fraction). Store at -80 C. 9. The pellet is the enriched mitochondrial fraction (or heavy membrane fraction). If intact mitochondria are desired, resuspend the pellet in 100 µl of Isotonic Mitochondrial Buffer (containing protease inhibitors). If mitochondrial protein lysate is desired, resuspend the pellet with 100 µl of the Mitochondrial Lysis Buffer containing protease inhibitors (Add protease inhibitor cocktail to Mitochondrial Lysis Buffer at 1:100 dilution before use). Store resuspended pellet at -80 C. 10. Take 20 µg of protein from each of the mitochondria and cytosol fractions and analyze by standard Western blotting methods. Use the antibodies at the following dilution ranges: Anti-Bcl2 0.5 µg/ml 2 µg/ml Anti-GAPDH 0.125 µg/ml 1 µg/ml Optimize as needed.
Sample Results Cyto Mito Cyto Mito 28 17 Bcl-2 38 GAPDH Figure 1. Mitochondria isolation from 293 cells. 293 cells were cultured with DMEM culture medium containing 10% FBS. Cells were harvested and processed according to the protocol using Millipore s Mitochondria/Cytosol Fractionation Kit (MIT1000). Mitochondria and cytosol fractions were analyzed by standard Western blotting methods. As shown, Bcl2 was detected in the Mitochondria fraction (Mito), where as GAPDH was localized to the cytosol fraction (Cyto). Related Products AP124P Goat anti-mouse IgG (H+L), Peroxidase-conjugated secondary antibody References 1. Jan. Y., et al. (2004). Cell, 116 : 751-762
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Cat No. MIT1000 Aug / 2010 Revision B