Key Words
Mitochondria Isolation, Discontinuous Sucrose Gradient, Large Volume
Pelleting, Saccharomyces cerevisiae, Superspeed Centrifuges,
Carbon Fiber Rotors
Introduction
Mitochondrial isolations are required for studies of a
wide range of biological questions. The purity of the
mitochondrial preparation is extremely important when
studying the functional assembly of mitochondrial DNA.
This procedure describes the isolation of mitochondria
from large volume cultures of Saccharomyces cerevisiae
using the Thermo Scientific Sorvall LYNX 6000
superspeed centrifuge. In addition, this procedure can be
readily adapted to other cell types, including bacteria,
insects, or mammalian cells.
The following protocols were developed based on
References 1–4.
Procedures
STEP 1: Pelleting the Mitochondria
1. Grow overnight a pre-culture of Saccharomyces
cerevisiae at 30 °C in Difco YM.
2. Transfer 8 mL of pre-culture into four flasks
containing 2 L of Difco YM and grow at 30 °C
overnight to OD600.
3. Place the culture into Thermo Scientific Nalgene
1 L superspeed bottles. Perform centrifugation in a
Thermo Scientific Fiberlite F9-6x1000 LEX or
F10-4x1000 LEX rotor and a Sorvall® LYNX 6000
superspeed centrifuge with the following parameters:
2,000 x g for 15 min at 4 °C.
4. Wash the pellet with ice-cold 18 ΩM H20 and
perform centrifugation with the following parameters:
2,000 x g for 15 min at 4 °C.
5. Re-suspend the pelleted cells in 0.1 M Tris-HCl, 1%
2-mercaptoethanol (pH 9.3), and incubate at 32 °C
for 10 min.
6. Re-pellet cells using 0.01 M Tris- HCl, 0.5 M KCl
(pH 7.0).
Figure 1. Thermo Scientific Sorvall LYNX 6000 superspeed
centrifuge with Fiberlite F9-6x1000 LEX rotor.
7. Re-suspend the cells in 0.01 M citrate-phosphate
buffer (pH 5.8), 1 mM EDTA, 1.35 M sorbitol.
8. Add 1 mg/mL Zymolyase 20T (ICN Biomedicals)
and gently shake for 90 min at 32 °C in N2
atmosphere to digest the cell wall for spheroplast
release.
9. Pellet the spheroplasts by performing centrifugation
in the Thermo Scientific Fiberlite F20-12x50 LEX
rotor and the Sorvall LYNX 6000 superspeeed
centrifuge with the following parameters: 3,000 x g
for 10 min at 4 °C.
Technical Note: TNCFGLYNXMITOCH 1012
Isolation of Mitochondria Using
the Thermo Scientific Sorvall
LYNX 6000 Superspeed Centrifuge
Alemania +49 6184 90 6000
Australia +61 39757 4300
Austria +43 1 801 40 0
Bélgica +32 53 73 42 41
China +86 21 6865 4588 or
+86 10 8419 3588
España/Portugal +34 93 223 09 18
SPANISH
Francia +33 2 2803 2180
Holanda +31 76 579 55 55
India +91 22 6716 2200
Italia +39 02 95059 554
Figure
Thermo
Japón
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45 The
453 9220
Nueva
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+64 9 980 6700
Scientific
Paises Nórdicos/Bálticos/CIS
F20-12x50 LEX rotor.
+358 9 329 10200
Rusiathe
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703 42 15
5.Collect
mitochondrial
pellet and re-suspend in
Suiza +41 44 454 12 22
pH UK/Irlanda
7.5 buffer.
Add
1%
SDS
and 50 mg/mL proteinase
+44 870 609 9203
USA/Canadáfor
+1 866
3766
K. Incubate
3 to9843.5
h at 37 °C with gently shaking.
Solution
can be
stored
Otros países
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+852 for
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4613in further studies.
Otros paises +49 6184 90 6000
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Conclusion
This technical note describes a process for the purification
of mitochondria from yeast cells using reliable Thermo
Scientific Fiberlite carbon fiber rotors with certified
biocontainment and dependable leakproof Thermo
Scientific
large
volume superspeed bottles. This
11.
Perform
centrifugation
with
the
following
© 2012 Thermo Fisher Scientific Inc. Tutti i diritti riservati. Tutti gli altri marchi sono di proprietà di Thermo Fisher
Scientific Inc.Nalgene
e delle sue filiali.
Specifiche,
condizioni
e tariffe possono subire
variazioni.
tutti i10
prodotti
sonoat
disponibili
in tutti
i paesi. Per1X.
maggiori dettagli
consultare il rivenditore
locale
combination,
along
with the Thermo Scientific Sorvall
parameters:
3,000
x gNonfor
min
4 °C.
Repeat
LYNX Svizzera
superspeed
centrifuge, offers the total solution
Australia
+61 39757 4300
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+91 of
22 6716
+41 44 454 12 22
12.
Re-suspend
the final pellet in 80
0.012200
M
for efficient
and+44
safe
of all your samples. This
Austria +43 1 801 40 0
Italia +39 02 95059 554
UK/Irlanda
870processing
609 9203
2 mM
BelgioTris-maleate,
+32 53 73 42 41 0.6 M mannitol,Olanda
+31EDTA,
76 579 55 55
USA/Canada +1 866 984 3766
protocol
can
be
adapted
to
retrieve
mitochondria from
Cina +86
21 6865
4588pH
or 6.8, at 4 °C and
Nuova
Zelanda
+64 9 980 6700
0.2%
BSA,
mix
thoroughly
+86 10 8419 3588
Europa del Nord/CIS
Altretypes
Nazioni
Asiatiche
+852 2885 4613
other cell
such
as bacterial,
mammalian, or insect cells.
glass
rod.
Franciawith
+33 2a2803
2180
+358 9 329 10200
Nazioni non in elenco +49 6184 90 6000
10. Discard the supernatant and suspend the pellet in
40 mL of 0.01 M Tris-maleate, 0.75 sorbitol, 0.4 M
mannitol 2 mM EDTA, 0.1%ITALIAN
BSA, pH 6.8.
thermoscientific.com
Germania +49 6184 90 6000
Russia +7 812 703 42 15
o +33 2 2803 2180
References
Giappone
+81 45 453
9220
Spagna/Portogallo
13.
Perform
centrifugation
with the
following +34 93 223 09 18
1. Guerin, B., Labbe, P., Somlo, M. (1979). Preparation of
parameters: 1,000 x g for 10 min at 4 °C.
yeast mitochondria (Saccharomyces cerevisiae) with
Collect the supernatant into clean tubes.
good P/O and respiratory control ratios. Methods
GERMAN
thermoscientific.com
14. Repeat steps 10-12 and combine supernatants.
Enzymol. 55, 149-159.
© 2012 Thermo Fisher Scientific Inc. Alle Rechte vorbehalten. Alle anderenWarenzeichen sind Eigentum von Thermo Fisher Scientific Inc. bzw. nationalen
15.
Perform
centrifugation
the und
following
A.,erhältlich.
Barrio,
E. (1990). A rapid and simple
Niederlassungen.
Änderungen
von Spezifikationen,with
Bedingungen
Preisen vorbehalten. Nicht alle Produkte2.
sindQuerol,
in allen Ländern
Ausführliche
Informationen
erhalten Sie bei17,000
Ihrer Thermo x
Fisher
Vertretung.
parameters:
g for
10 min at 4 °C.
method for the preparation of yeast mitochondrial
Collect the mitochondrial pellet.
DNA. Nucleic Acids Res. 18, 1657.
Australien +61 39757 4300
Belgien +32 53 73 42 41
Indien +91 22 6716 2200
Italien +39 02 95059 554
Deutschland Nationale kostenlose Hotline
Neuseeland +64 9 980 6700
Deutschland International
+358 9 329 10200
STEP
China +862:21Sucrose
6865 4588 or Gradient Purification
Japan +81 45 453 9220
+86Mitochondria
10 8419 3588
Niederlande +31 76 579 55 55
of
0800 1 536 376
Perform
the entire purification at 4Nordeuropa/Baltikum/CIS
°C.
+49 6184 90 6000
1 801 40
0
1.Prepare
a discontinuous sucroseÖsterreich
gradient+43
(30%
w/w,
Frankreich +33 2 2803 2180
40% w/w, 50% w/w, 60% w/w), using cold sucrose
solutions containing 1 mM EDTA, 0.1% BSA, 10 mM
Tris-HCl (pH 7.5)5.
Russland +7 812 703 42 15
Spanien/Portugal +34 93 223 09 18
3. Rickwood,
D.,44Wilson,
Schweiz +41
454 12 22 M. T., Darley-Usmar, V. M.
UK/Irland
+44 870and
609 9203
(1987).
Isolation
characteristics of intact
USA/Kanada +1 866 984 3766
mitochondria. In V. M. Darley-Usmar, D. Rickwood,
Andere asiatische
+852 2885 4613A Practical Approach
M. T.Wilson
(Ed.),Länder
Mitochondria:
Andere Länder +49 6184 90 6000
(pp. oder
1-16).
.
Oxford:
IRL
Press..
+33 2 2803 2180
4. Yaffe, M. P. (1991). Analysis of mitochondrial function
and assembly. Methods Enzymol. 194, 627-643.
FRENCH
5. Griffith, O.M. (2006). Practical Techniques for
thermoscientific.com
2.Re-suspend
a mitochondrial pellet in
Centrifugal Separations. In O.M. Griffith (Ed.),
© 2012
Thermo
Fisher
Inc. Tous
réservés. Les autres
marques
sont des marques commerciales ou déposées de Thermo Fisher
~ 0.5
mL
XScientific
number
ofdroits
centrifuge
of 0.5
Mdéposées
sucrose.
(pp.
Scientific Inc et de ses filiales. Les caractéristiques, conditions et tarifs sont susceptibles d’être modifiés. Tous les Fiberlite
produits ne sontCentrifuge
pas disponibles dans
tous 39-45).
les
pays. Pour tout renseignement,
vous adresser à votre
distributeur local. on the
3.Gently
layer theveuillez
mitochondrial
suspension
sucrose gradient and perform centrifugation in the
Allemagne +49 6184 90 6000
France +33 2 2803 2180
Russie +7 812 703 42 15
Fiberlite
F20-12x50
LEX rotor with
the22following
Australie
+61 39757
4300
Inde +91
6716 2200
Suisse +41 44 454 12 22
Autriche
+43 1 801 40
0
Italie
02 95059 554
UK/Irlande +44 870 609 9203
parameters:
51,000
x g for 2.5 hr
at+39
4 °C.
Belgique +32 53 73 42 41
Chine +86 21 6865 4588 or
Japon +81 45 453 9220
Nouvelle-Zélande +64 9 980 6700
+86Note:
a brown
band
10 8419The
3588 intact mitochondria form
Pays Bas
+31 76 579
55 55 at
Espagne/Portugal
+34 93 223density
09 18
Pays nordiques/baltes/CEI
1.19 g/mL sucrose
(approximately
the center of
+358 9 329 10200
the tube). One or more impurity bands may be seen
below the mitochondria band.
USA/Canada +1 866 984 3766
Autres pays d’Asie +852 2885 4613
Autres Pays +49 6184 90 6000
ou +33 2 2803 2180
thermoscientific.com/lynxsuperspeed
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consult your
localyour
saleslocal
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for details. for details.
terms
aresubject
subjecttotochange.
change.
all products
are available
in all countries.
consult
sales representative
Australia +61 39757 4300
Austria +43 1 801 40 0
Belgium +32 53 73 42 41
China +86 21 6865 4588 or
+86 10 8419 3588
France +33 2 2803 2180
Germany national toll free 0800 1 536 376
Germany international +49 6184 90 6000
TNCFGLYNXMITOCH 1012
India toll free 1800 22 8374
India +91 22 6716 2200
Italy +39 02 95059 554
Japan +81 45 453 9220
Netherlands +31 76 579 55 55
New Zealand +64 9 980 6700
Nordic/Baltic/CIS countries
+358 9 329 10200
Russia +7 812 703 42 15
Spain/Portugal +34 93 223 09 18
Switzerland +41 44 454 12 22
UK/Ireland +44 870 609 9203
USA/Canada +1 866 984 3766
Other Asian countries +852 2885 4613
Countries not listed +49 6184 90 6000
Technical Note: TNCFGLYNXMITOCH 1012
4.Remove the mitochondria band and dilute it with
2 volumes of 1 mM EDTA, 10 mM Tris HCl (pH 7.4).
Pellet the mitochondria by performing centrifugation
© 2012 Thermo Fisher Scientific Inc. Reservados todos los derechos. Las otras marcas comerciales son propiedad de Thermo Fisher Scientific Inc. y sus
with
the losfollowing
26,000 x g for 10 min
filiales. Características, condiciones y precios están sujetos a posibles cambios. Algunos productos no están disponibles
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