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

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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

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with their applicable instructions for a period of one year from
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Cat No. MIT1000

Aug / 2010
Revision B