Mitochondria/Cytosol Fractionation Kit

Transcription

1 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) Fax: +1 (951)

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3 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 ( UG): 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.

4 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 µg/ml 1 µg/ml Optimize as needed.

5 Sample Results Cyto Mito Cyto Mito 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 :

6 Warranty Millipore Corporation ( Millipore ) warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products. MILLIPORE MAKES NO OTHER WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty provided herein and the data, specifications and descriptions of Millipore products appearing in Millipore s published catalogues and product literature may not be altered except by express written agreement signed by an officer of Millipore. Representations, oral or written, which are inconsistent with this warranty or such publications are not authorized and if given, should not be relied upon. In the event of a breach of the foregoing warranty, Millipore s sole obligation shall be to repair or replace, at its option, the applicable product or part thereof, provided the customer notifies Millipore promptly of any such breach. If after exercising reasonable efforts, Millipore is unable to repair or replace the product or part, then Millipore shall refund to the Company all monies paid for such applicable Product. MILLIPORE SHALL NOT BE LIABLE FOR CONSEQUENTIAL, INCIDENTAL, SPECIAL OR ANY OTHER DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY COMPANY CUSTOMER FROM THE USE OF ITS PRODUCTS Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. Copyright Millipore Corporation 2010 All rights reserved

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8 Cat No. MIT1000 Aug / 2010 Revision B