A-1803A
A p p l i c a t i o n
I n f o r m a t i o n
High Performance Centrifugation
...............................................
Isolation of Mitochondria from Saccharomyces cerevisiae
Using the Avanti™ J High Performance Centrifuge
Anasuya S. Patel
Beckman Instruments, Inc.
Mitochondrial isolations are required for studies of a
wide range of biological questions, especially in
bioenergetics. The purity of the mitochondrial
preparations is extremely important when studying
functional assembly, and also for studies of mitochondrial DNA. Procedures have been continuously
refined to provide the best purities possible. Such
isolation protocols generally call for isopycnic centrifugation at forces as high as 80,000 × g. We
present here an optimized procedure for isolation of
mitochondria in which this high-force, isopycnic
centrifugation is performed without an ultracentrifuge. The Beckman Avanti J is a high performance
centrifuge with a switched reluctance drive that can
generate forces sufficient for the isopycnic separation.
The procedure presented below describes the
isolation of mitochondria from the yeast Saccharomyces cerevisiae, but can be readily adapted to other
cell types. The following protocols were developed
based on References 1–4.
Protocols
Pelleting the Mitochondria
1.
2.
3.
4.
5.
Grow up overnight a preculture of the yeast
Saccharomyces cerevisiae at 30°C in Difco
YM broth.
Transfer 8 mL of preculture into four flasks
containing 2 L of the above medium and grow
cells at 30°C overnight to OD600 ≅ 3.0. Pellet
the cells at 2000 × g for 15 min.
Wash the cells with cold distilled water. The
Beckman J-6 centrifuge with either the JS-4.2
or JS-5.2 rotor accommodating adapters to spin
250-mL conical bottles will serve the purpose.
Pellet the cells at 2000 × g for 15 min.
Suspend the washed cells in 0.1 M Tris-HCl,
1% 2-mercaptoethanol, pH 9.3, and incubate at
32°C for 10 min.
Wash the cells thoroughly using 0.01 M TrisHCl, 0.5 M KCl, pH 7.0. About 3 L of the
washing buffer is required for 20.0 g wet
weight.
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6.
Suspend washed cells in 0.01 M citrate-phosphate buffer, pH 5.8, 1 mM EGTA, 1.35 M
sorbitol. Usually 40 mL are required for 20.0 g
wet weight.
7. Add 1 mg/mL Zymolyase 20T (ICN
Biomedicals) and shake the mixture at low
speed for 90 min at 32°C in N2 atmosphere
(zymolyase digests the cell wall, releasing
spheroplasts).
8. Centrifuge the spheroplast suspension for
10 min at 5000 × g at 4°C. Discard the supernatant.
9. Wash the pellet twice with 40 mL of 0.01 M
Tris-maleate, 0.75 sorbitol, 0.4 M mannitol,
2 mM EGTA, 0.1% BSA, pH 6.8.
10. Suspend the final pellet in 80 mL of 0.01 M
Tris-maleate, 0.6 M mannitol, 2 mM EGTA,
0.2% bovine serum albumin, pH 6.8, at 4 °C.
Mix thoroughly with a glass rod.
11. Centrifuge at 800 × g for 10 min. Collect the
supernatant. Repeat step 10 and combine supernatants. Centrifuge at 12,000 to 15,000 × g
for 10 min, 4°C, in the Avanti JA-25.50 rotor.
Collect the mitochondrial pellet.
Figure 1. Purification of mitochondria by sucrose
density gradient centrifugation in the JA-25.50 rotor.
Sucrose Gradient Purification of the
Mitochondria in the JA-25.50 Rotor
The entire purification must be carried out at 4°C.
1. Prepare a 1–2 M (34–70% wt/vol) sucrose gradient containing 1 mM EDTA, 0.1% BSA,
10 mM Tris-HCl, pH 7.5. It can be a continuous gradient prepared using a gradient mixer or
a step gradient prepared by any convenient
method. Cool the gradient to 4°C.
2. Thoroughly and gently resuspend the mitochondrial pellet in about 0.5 mL of 0.5 M sucrose buffer.
3. Gently layer the mitochondrial suspension over
the sucrose gradient and centrifuge for 2 h in
the JA-25.50 rotor at 75,600 × g (25,000 rpm).
The intact mitochondria form a brown band at
≅ 1.19 g/mL sucrose density. One or more impurity bands may be seen below the main band.
4. Remove the main mitochondrial band and dilute it with 2 volumes of 1 mM EDTA,10 mM
Tris-HCl, pH 7.4 buffer. Pellet the pure mitochondria by centrifugation at 20,000 × g for
10 min (this is 13,000 rpm in the JA-25.50 rotor).
5. Collect the mitochondrial pellet. Figure 1
shows a typical mitochondrial band separation
in a sucrose gradient using the Avanti J centrifuge.
Characterization by DNA Extraction
The mitochondrial isolate may be characterized by
pelleting the organelle DNA and analyzing with a
restriction enzyme such as EcoRV. A procedure for
this is given below.
1. Resuspend mitochondria in 100 mL of 10 mM
Tris-HCl, 1 mM EDTA and 50 mM NaCl,
pH 7.5 buffer. Add 1% SDS and 50 µg/mL
proteinase K. Incubate for 3 to 3.5 h at 37°C
with slow shaking.
2. Cool in ice, then add 25 mL of 5 M potassium
acetate. Incubate at 0°C for about 45 min.
3. Centrifuge at 5000 × g for 10 min at 4°C. Collect the supernatant and add one volume of
isopropanol. Mix and freeze at -20°C for
60 min.
4. Centrifuge at 10,000 × g for 10 min. Collect
the DNA precipitate.
5. Wash the DNA with 70% ethanol and vacuum
dry.
6. Treatment of the mtDNA with the restriction
nuclease, EcoRV (Gibco BRL), can be performed according to the protocol supplied by
the supplier.
2
7.
8.
Carry out the DNA digestion for 1 h at 37 °C
Stop the reaction with EDTA by adjusting the
concentration to 10 mM using 0.5 M EDTA,
pH 8.0.
Electrophorese the enzyme digest on 1.2% agarose gel using 0.04 M Tris-acetate, 0.001 M
EDTA, pH 8.0 buffer. Use 0.5 µg/mL ethidium
bromide in the agarose gel to visualize the
DNA bands. A typical electrophoresis pattern
is shown below in Figure 2.
2.
3.
4.
References
1.
Guerin, B., Labbe, P., Somlo, M. Preparation
of yeast mitochondria (Saccharomyces
cerevisiae) with good P/O and respiratory control ratios. Methods Enzymol. 55, 149-159
(1979)
Querol, A., Barrio, E. A rapid and simple
method for the preparation of yeast mitochondrial DNA. Nucleic Acids Res. 18, 1657
(1990)
Rickwood, D., Wilson, M. T., Darley-Usmar,
V. M. Isolation and characteristics of intact
mitochondria. Mitochondria: A Practical
Approach, pp. 1-16. Edited by V. M. DarleyUsmar, D. Rickwood, and M. T. Wilson.
Oxford, IRL Press, 1987.
Yaffe, M. P. Analysis of mitochondrial function and assembly. Methods Enzymol. 194,
627-643 (1991)
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Figure 2. Agarose gel electrophoresis of EcoRV-digested Saccharomyces cerevisiae mtDNA.
Lane 1: Molecular marker λ DNA Bstll digest; Lanes 2-5: EcoRV-digested mtDNA at various concentrations.
3
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