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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.
Millipores 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 g
5. Anti-GAPDH (CS204254): 25 g

Storage
All components should be store at -20C for up to o ne year from
date of receipt.

Assay Instructions
1. Culture cells in 10 cm tissue culture dishes until confluent
(~2x 107cells 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 4C 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
4C.
8. Collect the supernatant (cytosol and microsome fraction light membrane fraction). Store at -80C.
9. The pellet is the enriched mitochondrial fraction (or heavy
membrane fraction). If intact mitochondria are desired,
resuspend the pellet in 100 L of Isotonic Mitochondrial
Buffer (containing protease inhibitors). If mitochondrial
protein lysate is desired, resuspend the pellet with 100 L
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 -80C.
10. Take 20 g 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 g/mL 2 g/mL
Anti-GAPDH 0.125 g/mL 1 g/mL
Optimize as needed.

Sample Results
Cyto

Cyto Mito

28

Mito

38

GAPDH

Bcl-2

17

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
Millipores 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

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 Millipores 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, Millipores 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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Cat No. MIT1000


Aug / 2010
Revision B