Mitochondrial Fractionation Kit
For the isolation of mitochondrial & cytosolic fractions(version C2)

Catalog No. 40015

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USA
Toll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone: 0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:
+81 3 5261 8733
Copyright 2009 Active Motif, Inc.

www.activemotif.com

Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any
kind, either explicit or implied. Information may be changed or updated in this manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation isproprietary information and protected by the copyright laws of the United States and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2009 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respectivecompanies.
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TABLE OF CONTENTS

Page

Overview  . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Example Experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
ProtocolGeneral Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Buffer Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Cell Fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
AppendixSection A. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Section B. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

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Overview
The Mitochondrial Fractionation Kit was designed to facilitate the study of apoptotic and signaltransduction pathways. It provides unique formulations of buffers and reagents that enable
isolation of highly enriched mitochondrial and cytosolic fractions from various mammalian celllines, including both apoptotic and non-apoptotic cells. The fractionation procedure is simple
and easy to perform. No ultra-centrifugations are required and no toxic chemicals are involved.
The enriched fractions can be used to study translocation of a variety of proteins between themitochondrial and cytosolic fractions by Western blot analysis, ELISA or other assays.
Lysis is performed in a pestle homogenizer using the gentle Cytosolic Buffer. This helps toeliminate accidental rupture of the mitochondria and the subsequent leakage of mitochondrial
proteins (such as cytochrome c) into the cytosolic fraction. After lysis, the different cellularcompartments are separated by centrifugation of the cellular lysate at different speeds to isolate,successively, the nuclei, mitochondria and cytosol.

product

formatcatalog no.

Mitochondrial Fractionation Kit

100 rxns

40015

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1

Example Experiment
To determine the mitochondrial and cytosolic cytochrome c levels, 5 x 107 HeLa cells were grown
to 90-95% confluence. The cells were harvested and mitochondrial and cytosolic fractions wereisolated using the Mitochondrial Fractionation Kit. Active Motif’s FunctionELISA™ Cytochrome c Kit,
which has since been discontinued, and Western blot analysis were used to monitor and quantify
mitochondrial and cytosolic cytochrome c (Figure 1).
1FunctionELISA Cytochrome c23451250 kDa –0.75
Cyto 1

98 kDa –64 kDa –A 450

Cyto 20.5
Mito 150 kDa –Mito 236 kDa –30 kDa –

0.25

A0B

HeLa
Unstimulated Extracts (2 ug/well)

16 kDa –6 kDa –
4 kDa –

Figure 1: Location of cytochrome c in HeLa cells.
Mitochondrial and cytosolic extracts were isolated from HeLa cells grown to 90~95% confluence using the Mitochondrial
Extract Kit. Five µg of each lysate was tested in duplicate using the FunctionELISA Cytochrome c Kit (Fig. 1A). 25 µg of eachlysate was tested in duplicate by Western blot analysis using a 4-20% Tris-Glycine gel (Fig. 1B).
Lane 1: Protein standard
Lane 2: Cytosolic extract, sample 1
Lane 3: Cytosolic extract, sample 2
Lane 4: Mitochondrial extract, sample 1
Lane 5: Mitochondrial extract, sample 2

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2

Kit Components and Storage
Please store each component at the temperature indicated in the table below. Do not re-freeze
the Protein G Magnetic Beads.
Reagents

Quantity

Storage / Stability

1X Cytosolic Buffer
110 ml		

4°C for short term-20°C for 6 months

Mitochondria Buffer

10 ml

4°C for 6 months

1 M DTT

100 µl

-20°C for 1 year

Protease Inhibitor Cocktail

100 µl

-20°C for 1 year

Note:

The included buffers and reagents have been optimized for use with this kit.
Substitution with other reagents may not give optimal results.

Additional materials required• Cell scraper• Phosphate buffered saline (PBS, pH 7.4)
• Pestle homogenizer• Centrifuge• Sterile, 15 ml centrifuge tubes• Microcentrifuge• Microcentrifuge tubes• Pipettors and pipette tips
• Bio-Rad assay reagent and BSA standard, or similar• 96-well, clear, flat-bottomed plate• Microplate reader)www.activemotif.com

3

Protocols
PLEASE READ THE ENTIRE PROCEDURE BEFORE STARTING!
General Considerations• Store all kit components as recommended• Keep buffers and sample on ice at all times during the procedure• The number of strokes required to lyse the cells with a pestle homogenizer but leave themitochondria intact depends on the cell line. For HeLa, PC3, DU145, JCA1 and ALVA31 cell lines,
between 30 and 50 strokes seem to work well. In contrast, mitochondria from Jurkat andLNCaP cells appear to be more fragile and only 30 or fewer strokes should be applied. You
may need to optimize the lysis procedure for your given cell line and/or treatment.

Buffer Preparation
Preparation of Complete Mitochondria Buffer
Prepare the amount of Complete Mitochondria Buffer required for your experiment by adding
2 µl Protease Inhibitor Cocktail and 1 µl DTT per 1 ml of Mitochondria Buffer. Some of the proteaseinhibitors lose their activity within 24 hours of dilution. Therefore, we recommend using theComplete Mitochondria Buffer immediately. Any remaining amount should be discarded if notused in the same day.

Cell Fractionation
This procedure is to be used for fractionation of ~5 x 107 cells.
1.

Wash the cells 1X with 10 ml ice-cold PBS.

2.

Add 10 ml ice-cold PBS and scrape the cells off the dish using a cell scraper. Transfer the cells
to a pre-chilled 15 ml centrifuge tube and spin at 600 x g for 5 minutes at 4°C.

3.

Remove supernatant and wash cell pellet 1X by gently resuspending it in 5 ml ice-cold PBS.

4.

Spin at 600 x g for 5 minutes at 4°C and remove supernatant.
Note:

For apoptosis-induced cells, detachment of dead cells during the wash step
will reduce the number of cells (relative to a plate of “normal” cells) that are
obtained by scraping the plate. Therefore, to obtain fractions of an adequate
concentration, buffer volumes may need to be reduced to coincide with thereduced number of cells being fractionated.

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45.

Add 1 ml ice-cold 1X Cytosolic Buffer. Resuspend the cell pellet by pipetting up and down
gently with a pipette. Incubate on ice for 15 minutes, then transfer to a pre-chilled pestle
homogenizer.

6.

On ice, homogenize the cells using 30-50 strokes with the homogenizer. Transfer thesupernatant to a pre-chilled microcentrifuge tube.

7.

Spin the lysate at 800 x g (~3000 rpm) for 20 minutes at 4°C. The resulting pellet is the nuclei,cellular debris and intact cells, whereas the supernatant contains the cytosol, including themitochondria.

8.

Transfer the supernatant to a fresh, pre-chilled microcentrifuge tube.

9.

Spin the supernatant a 2nd time at 800 x g (~3000 rpm) for 10 minutes at 4°C to remove any
residual nuclei.

10. Transfer the supernatant to a fresh, pre-chilled microcentrifuge tube.
11.

Spin the supernatant at 10,000 x g (~11,000 rpm) for 20 minutes at 4°C to pellet themitochondria. The supernatant is the cytosolic fraction.

12.

Transfer the supernatant to a fresh pre-chilled microcentrifuge tube.

13.

Wash the mitochondrial pellet 1X with 100 µl 1X Cytosolic Buffer and spin at
10,000 x g for 10 minutes at 4°C. Remove and discard the supernatant.

14.

Lyse the mitochondrial pellet by adding 100 µl Complete Mitochondria Buffer and incubating
on ice for 15 minutes. After incubation, vortex for 10 seconds to mix thoroughly. This is theMitochondrial Fraction.

15.

Centrifuge the cytosolic supernatant obtained in step 12 at 16,000 x g (~14,000 rpm) for atleast 20 minutes at 4°C to remove any residual mitochondria. Transfer the cytosolic
supernatant to a fresh, pre-chilled microcentrifuge tube. This is the Cytosolic Fraction.

16.

Measure the protein concentration of each fraction using a Bio-Rad assay with a BSA
standard curve, or similar.

17.

Aliquot the fractions and store at -80°C. Avoid freeze/thaw cycles.

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5Appendix
Section A. Troubleshooting Guide
Problem/question

Possible Cause

Recommendation

Low protein
concentration incytosolic fraction

Cell lysis was not efficient

Increase the number of strokes performed withthe homogenizer

Mitochondrial proteinleakage

Cell lysis was too vigorous

Reduce the number of strokes with thehomogenizer to 30 strokes

Low protein
concentration in
mitochondrial fraction

Complete Mitochondria Buffer not madeproperly

Add 2 µl Protease Inhibitor Cocktail and1 µl DTT per 1 ml of bufferwww.activemotif.com

6Section B. Related Products
Transcription Factor ELISAs		FormatCatalog No.

TransAM™ c-Myc		
1 x 96 rxns
43396
TransAM™ MAPK Family		
2 x 96 rxns
47296
For a complete list of the over 40 TransAM Kits available, please visit www.activemotif.com/transam
in-cell Western Phospho ELISAs		FormatCatalog No.

FACE™ Bad		
1 x 96 rxns
48165
FACE™ FAK		
1 x 96 rxns
48145
FACE™ JNK		
1 x 96 rxns
48110
For a complete list of the over 20 FACE Kits available, please visit www.activemotif.com/face
Cell Extracts		

FormatCatalog No.

HeLa nuclear extract		
HeLa nuclear extract (TNF-a stimulated)		
HeLa whole-cell extract (TNF-a stimulated)		
Nuclear Extract Kit		

200 µg200 µg200 µg100 rxns

36010
40210
40200
40010

Apoptosis Assays		

FormatCatalog No.

DNA Damage Assay (Fluorescent)		
Mitochondrial Fractionation Kit		

2 x 96 rxns
100 rxns

18030
48015

Apoptosis-related Antibodies

FormatCatalog No.

Application

Bcl-10 mAb (Clone 151)
WB
200 µg39393
Caspase-3 mAb
WB
100 µg40924
Caspase-7 mAb
WB
100 µg40929
Caspase-8 mAb
FACS, IHC, WB
100 µg40930
Caspase-9 pAb (Pro-form)
WB
100 µg40931
Caspase-14 mAb
WB
100 µg40932
D4-GDI mAb
IP, WB
100 µg
40941
DAP-3 mAb
WB
100 µg
40963
DAP-5 mAb
WB
100 µg40964
DcR3 mAb
WB
100 µg40933
DR4 mAb
FACS, WB
100 µg40934
DR6 mAbWB
100 µg40927
DRAK1 pAb
WB
100 µg40923
DRAK2 pAb
WB
100 µg40926
Livin/ML-IAP mAb
WB
100 µg40958
MyD88 pAb
WB
100 µg40914
PARP-1 N-terminal pAb
WB
200 µl39559
PARP-1 C-terminal pAb
WB
200 µl39561
Rad21 mAb (Clone 52A311)
WB
200 µg
39383
STAT3 phospho Tyr705 pAb
WB
200 µl39595
TRAF2 mAb
WB
100 µg40919
TRAF5 mAb
WB
100 µg40920
TRAIL mAb
FACS, WB
100 µg40966
For an up-to-date list of Active Motif’s nearly 400 antibodies, please visit www.activemotif.com/abs
Application Key: ChIP = Chromatin Immunoprecipitation; EMSA = Electrophoretic Mobility Shift Assay;
IF = Immunofluorescence; IHC = Immunohistochemistry; IP = Immunoprecipitation; WB = Western blot

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7Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USAToll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone: 		+32 (0)2 653 0001
Fax: 		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
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