CHAPTER

Isolation of mitochondria
from cells and tissues

Pin-Chao Liaoa,†, Christian Bergaminib,†, Romana Fatob, Liza A. Pona,∗

1
Francesco Pallottic,∗
a
Department of Pathology and Cell Biology, Columbia University, New York, NY, United States
b
Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
c
Department of Medicine and Surgery, University of Insubria, Varese, Italy
∗
Corresponding authors: e-mail address: [email protected];
[email protected]

Chapter outline
1 Isolation of mitochondria from rat brain..................................................................5
1.1 Definition............................................................................................5
1.2 Rationale............................................................................................6
1.3 Materials, equipment and reagents.........................................................6
1.3.1 Reagents..........................................................................................6
1.3.2 Reagents for gradients.......................................................................6
1.3.3 Equipment........................................................................................6
1.4 Protocols.............................................................................................7
1.4.1 Preparation of two-step discontinuous gradients.................................7
1.4.2 Preparation of crude mitochondria from rat brain................................7
1.4.3 Preparation of synaptonemal LM and HM fractions and free
mitochondria from crude mitochondria...............................................8
1.5 Analysis and statistics..........................................................................8
1.6 Related techniques...............................................................................9
1.7 Pros and cons......................................................................................9
1.8 Troubleshooting and optimization...........................................................9
2 Isolation of mitochondria from rat liver.................................................................10
2.1 Definition..........................................................................................10
2.2 Rationale..........................................................................................10
2.3 Materials...........................................................................................10
2.4 Equipment........................................................................................10
2.5 Protocols...........................................................................................11

†
P.-C.L. and C.B. contributed equally.

Methods in Cell Biology, Volume 155, ISSN 0091-679X, https://doi.org/10.1016/bs.mcb.2019.10.002

© 2020 Elsevier Inc. All rights reserved.
3
4 CHAPTER 1 Isolation of mitochondria from cells and tissues

2.6 Safety considerations and standards.....................................................11

2.7 Analysis and statistics........................................................................11
2.8 Related techniques............................................................................11
2.9 Pros and cons....................................................................................12
2.10 Alternative methods/procedures...........................................................12
2.11 Troubleshooting and optimization.........................................................12
3 Isolation of mitochondria from beef heart..............................................................13
3.1 Definition..........................................................................................13
3.2 Rationale..........................................................................................13
3.3 Materials...........................................................................................13
3.4 Working solutions...............................................................................13
3.5 Equipment........................................................................................13
3.6 Protocol............................................................................................14
3.7 Analysis and statistics........................................................................14
3.8 Related techniques............................................................................15
3.9 Pros and cons....................................................................................15
3.10 Troubleshooting and optimization.........................................................15
4 Isolation of mitochondria from skeletal muscle......................................................16
4.1 Definition..........................................................................................16
4.2 Rationale..........................................................................................16
4.3 Materials, equipment and reagents.......................................................16
4.4 Protocols...........................................................................................17
4.5 Related techniques............................................................................17
4.6 Pros and cons....................................................................................18
4.7 Alternative methods/procedures...........................................................18
4.8 Troubleshooting and optimization.........................................................18
5 Isolation of mitochondria from cultured cells.........................................................18
5.1 Definition..........................................................................................18
5.2 Rationale..........................................................................................19
5.3 Materials...........................................................................................19
5.4 Protocol............................................................................................19
5.5 Analysis and statistics........................................................................20
5.6 Related techniques............................................................................20
5.7 Pros and cons....................................................................................20
5.8 Alternative methods/procedures...........................................................20
5.9 Troubleshooting and optimization.........................................................21
6 Affinity purification of mitochondria from yeast using magnetic beads.....................21
6.1 Definition..........................................................................................21
6.2 Rationale..........................................................................................21
6.3 Materials, equipment and reagents.......................................................23
6.4 Materials...........................................................................................23
6.5 Equipment........................................................................................23
6.6 Protocols...........................................................................................24
 1 Isolation of mitochondria from rat brain 5

6.7 Analysis and statistics........................................................................25

6.7.1 Examination of mitochondrial purity.................................................25
6.8 Characterization of mitochondrial integrity............................................25
6.9 Related techniques............................................................................26
6.10 Pros and cons....................................................................................26
6.11 Troubleshooting and optimization.........................................................26
Acknowledgments....................................................................................................27
References..............................................................................................................27

Abstract
Isolated mitochondria are useful to study fundamental processes including mitochondrial res-
piration, metabolic activity, protein import, membrane fusion, protein complex assembly, as
well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs, and other
organelles. In addition, studies of the mitochondrial proteome, phosphoproteome, and lipi-
dome are dependent on preparation of highly purified mitochondria (Boldogh, Vojtov,
Karmon, & Pon, 1998; Cui, Conte, Fox, Zara, & Winge, 2014; Marc et al., 2002;
Meeusen, McCaffery, & Nunnari, 2004; Reinders et al., 2007; Schneiter et al., 1999;
Stuart & Koehler, 2007). Most methods to isolate mitochondria rely on differential centrifu-
gation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue
debris, and nuclei from whole cell extracts followed by high speed centrifugation to concen-
trate mitochondria and separate them from other organelles. However, methods to disrupt cells
and tissue vary. Moreover, density gradient centrifugation or affinity purification of the organ-
elle are used to further purify mitochondria or to separate different populations of the organ-
elle. Here, we describe protocols to isolate mitochondria from different cells and tissues as
well as approaches to assess the purity and integrity of isolated organelles.

1 Isolation of mitochondria from rat brain

1.1 Definition
Mitochondria from even a single region of the brain are highly heterogeneous based on
their morphological, histochemical, and enzymatic characteristics. Most methods are
designed to isolate three distinct populations of mitochondria from rat brain: (a) non-
synaptic mitochondria, the so-called “free mitochondria” (FM); (b) synaptosomal
mitochondria (synaptic), which can be further subdivided into two fractions based on
sedimentation properties, heavy (HM) and light (LM) (Reijnierse, Veldstra, & Van
den Berg, 1975; Van den Berg, 1973). Synaptosomal mitochondria are involved in
regulating neurotransmitter release and synaptic vesicle formation. In contrast,
non-synaptic mitochondria derive from multiple cell types and from neuronal soma
and are involved in microRNA regulation and energy production (Ly & Verstreken,
2006; Vos, Lauwers, & Verstreken, 2010; Wang, Sullivan, & Springer, 2017).
6 CHAPTER 1 Isolation of mitochondria from cells and tissues

1.2 Rationale
The starting material for this preparation is usually dissected rat or mouse brain,
sometimes pooled in three or four units from the same strain. Crude mitochondria
are isolated from brain extracts by differential centrifugation. Different populations
of mitochondria are prepared from crude mitochondria using discontinuous gradients
(sucrose, Ficoll, or metrizamide).

1.3 Materials, equipment and reagents

1.3.1 Reagents
The pH of all solutions is checked daily.

Solution A: Sucrose 0.32 M, EDTA-K+ 1.0 mM, Tris–HCl 10 mM, pH 7.4

Solution B: Sucrose 0.32 M, EDTA-K+ 50 μΜ, Tris–HCl 10 mM, pH 7.4
Solution C: Tris–HCl 6 mM, pH 8.1
Solution D: Mannitol 0.24 M, Sucrose 60 mM, EDTA-K+ 50 μΜ, Tris–HCl
10 mM, pH 7.4
Solution E: Ficoll 3% (w/w), Mannitol 0.12 M, Sucrose 30 mM, EDTA-K+
25 μΜ, Tris–HCl 5 mM, pH 7.4
Solution F: Mannitol 0.22 M, Sucrose 0.07 M, Tris–HCl 50 mM, EDTA 1 mM,
pH 7.2

1.3.2 Reagents for gradients

Gradient I:
Ficoll 7.5% (w/w) in solution B
Ficoll 12% (w/w) in solution B
Gradient II:
Ficoll 4.5% (w/w) in solution D
Ficoll 6% (w/w) in solution D

1.3.3 Equipment
For tissue homogenization
Potter-Elvehjem homogenizer consisting of a large clearance (0.1–0.15 mm,
volume 10 mL) Teflon pestle driven into a glass vessel mechanically (Braun
S homogenizer)
For differential centrifugation
Centrifuge: Beckman J2-21 or Sorvall RC-5B
Fixed Angle Rotor: Beckman JA-20 or Sorvall SS-34
Tubes: Polyallomer thick-wall, 10 mL
For gradients
Ultracentrifuge: Sorvall OTD65B or Beckman L5-50
Swinging bucket rotor: AH-650 or SW 50.1
Tubes: Polyallomer thin-wall, 5 mL
 1 Isolation of mitochondria from rat brain 7

1.4 Protocols
1.4.1 Preparation of two-step discontinuous gradients
Solutions for gradients are prepared just before the use and kept on ice. Gradients are
prepared just before the use and kept in the refrigerator.

Gradient I:
1. Place 1.9 mL of the 12% Ficoll solution in a 5 mL ultracentrifuge tube.
2. Gently place 1.9 mL of the Ficoll 7.5% step on top of the 12% Ficoll step,
using caution to avoid mixing of the steps.
3. Store at 4 °C.
Gradient II:
1. Place 1.4 mL of the 6% Ficoll solution in a 5 mL ultracentrifuge tube.
2. Gently place 2.6 mL of the 4.5% Ficoll solution on top of the 6% Ficoll step,
using caution to avoid mixing of the steps.
3. Store at 4 °C.

1.4.2 Preparation of crude mitochondria from rat brain

All solutions used are at 0–4 °C. Each step of homogenization is performed at 0–4 °C.

1. The procedure described below is based on preparation from one rat brain (ca.
120 mg). Rats are sacrificed using established guidelines of ethical procedures.
2. Place rat brain in a prepared refrigerated box (0–4 °C) within 15 s of
euthanization and separate hemicortexes from hippocampus and striatum.
3. Rinse and place the hemicortexes (freed from blood and debris) in 2.5 mL of
solution A.
4. Homogenize the tissue in a Teflon-glass homogenizer by five up-and-down
passes of the pestle at 800 rpm for a maximum of 1 min.
5. Transfer the homogenate to a pre-chilled centrifuge tube, resuspend residual cell
extract in the homogenizer in 2.5 mL of solution A and transfer this material to
the centrifuge tube.
6. Centrifuge the homogenate by gradually increasing the centrifugation rate to
1000  g over a period of 4 min, with centrifugation at 1000  g for additional
11 s. Transfer the supernatant to a fresh centrifuge tube and store on ice.
7. Resuspend the pellet with 2 mL of solution A and homogenize the resuspended
pellet at 300 rpm for 60 s and 3 passages of the pestle, and transfer the
homogenate to a fresh centrifuge tube.
8. Resuspend residual cell extract in the homogenizer in 2.0 mL of solution A and
transfer this material to the centrifuge tube containing the homogenate from
step 7.
9. Centrifuge the homogenate as in steps 6–7 and pool the supernatant with the
supernatant from the first round of homogenization.
10. Carry out one more round of homogenization of the pellet as in steps 8–9.
11. Centrifuge the three pooled supernatants at 15,000  g for 20 min.
12. The pellet obtained is crude mitochondria.
8 CHAPTER 1 Isolation of mitochondria from cells and tissues

1.4.3 Preparation of synaptonemal LM and HM fractions and free

mitochondria from crude mitochondria
1. Discard the supernatant and resuspend the “crude” mitochondrial pellet from
step 12 above (which contains non-synaptic and synaptic mitochondria) to a
final volume of 0.7 mL in solution A and homogenize at 150 rpm and 3 passages
of the pestle.
2. Gently apply the suspension to gradient I using caution to avoid mixing the
gradient steps.
3. Centrifuge in a swinging bucket rotor at 73,000  g for 24 min. Myelin and
synaptosomal mitochondria are recovered in two bands. The synaptosomal
mitochondrial band is at the interface of the 7.5–12% gradients. Free
mitochondria (FM) are recovered in the pellet.
4. Discard the myelin band and recover the synaptosomal band with a Pasteur
pipette and place in a centrifuge tube.
5. Dilute the synaptonemal band with solution A to a final volume of 5 mL and
centrifuge at 15,000  g for 20 min.
6. To isolate free mitochondria (FM), resuspend the pellet obtained from gradient
I in solution F to a final volume of 0.6–0.8 mL. This is the final FM fraction.
7. To isolate HM and LM fractions, resuspend the pellet obtained from step 5 in
5 mL of solution C and homogenize at 150 rpm for 2 min and four pestle strokes.
8. Transfer the homogenate to a pre-chilled centrifuge tube and centrifuge the
suspension at 14,000  g for 30 min.
9. Resuspend the pellet in 5 mL of solution C and centrifuge the suspension at
14,000  g for 30 min.
10. Resuspend the pellet in solution E to a final volume 0.6 mL.
11. Gently apply the suspension to gradient II, using caution to avoid mixing.
12. Centrifuge at 10,000  g for 30 min. The HM fraction is in the pellet and the LM
fraction is at the interface of the 4.5–6% steps.
13. To obtain the LM fraction, discard the band at the interface of the 3–4.5% steps.
Transfer the LM fraction in band at the 4.5–6% interface to a fresh centrifuge
tube with a Pasteur pipette and dilute it in an equal volume of solution A.
14. Centrifuge the suspension at 15,000  g for 30 min.
15. Discard the supernatant and resuspend the pellet in solution F to a final volume
of 0.6–0.8 mL. This is the final LM fraction.
16. To obtain the HM fraction, resuspend the pellet obtained from gradient II in
solution F to a final volume of 0.6–0.8 mL. This is the final HM fraction.
17. Store all fractions at 80 °C.

1.5 Analysis and statistics

Protein content is determined by the Lowry method (Lowry, Rosebrough, Farr, &
Randall, 1951). Expected yields of mitochondrial protein from different fractions
are: 5 mg/mL for FM; 1.50 mg/mL for LM; 1.60 mg/mL for HM, corresponding
 1 Isolation of mitochondria from rat brain 9

to 10.8% (FM), 2.0% (LM), and 2.4% (HM) of the homogenate protein. The
method allows for the preparation of highly purified mitochondrial fractions from
cerebral cortex of rat brain suitable for respiratory chain enzymatic activities
(Pallotti & Lenaz, 2007). To ensure NADH availability, mitochondrial fractions
are usually pulse sonicated 5 times for 10 s/min at 150 W in an ice bath under
nitrogen gas prior to measurement of enzymatic activity (Pallotti et al., 1998).
Cytochrome content can be evaluated by using the method of Vanneste (1966)
and Nicholls (1976). These mitochondrial preparations can be assayed for CoQ
content by reversed phase HPLC analysis after extraction with methanol and light
petroleum (Battino et al., 1995).

1.6 Related techniques

Some recent methods can be applied for the isolation of mitochondria from an entire
hemisphere of neonatal mice (Wang et al., 2011). Recent methods have been published
for isolation of mitochondria from postmortem human brain for mtDNA analysis
(Devall et al., 2015) and for isolation of mitochondria in CNS injury and neurodegen-
eration (Hubbard et al., 2019), using magnetic beads that are conjugated to antibodies
against TOM22.

1.7 Pros and cons

Pros Cons

Highly purified mitochondria Relatively low

yield
Conservation of biochemical and functional properties Time intensive
Allows for isolation of different populations of mitochondria, increasing
selectivity and sensibility
High reproducibility

1.8 Troubleshooting and optimization

Problem Solution

Low yield Repeat homogenization of the low speed

(1000  g) pellet.
Altered specific enzymatic activities Wash the “crude” mitochondrial pellet least 2–3
(markers of subcellular fractions) times to remove the microsomal contamination.
10 CHAPTER 1 Isolation of mitochondria from cells and tissues

2 Isolation of mitochondria from rat liver

2.1 Definition
Rat liver is a suitable source of mitochondria, as it is easily obtained and less difficult
to homogenize compared to other tissues. Moreover, the yield of mitochondria is typ-
ically high because the hepatocytes account for 90% of the liver volume and are rich
in mitochondria (about 1000 per cell; 20% of the total cellular protein). Mitochondria
isolated from rat liver (RLM) maintain a high degree of coupling for more than 3 h
after isolation and are normally used for biochemical studies, including polaro-
graphic measurements.

2.2 Rationale
To obtain the RLM, we followed the differential centrifugation method of Costantini,
Petronilli, Colonna, and Bernardi (1995) with some modifications. Rats are starved
during the night before the experiment to lower endogenous fatty acid levels and the
glycogen content of the liver. Animals are sacrificed and the liver is immediately
transferred to ice-cold extraction buffer. The extraction buffer contains albumin to
remove free fatty acids, which uncouple oxidative phosphorylation. The tissue is
then minced with scissors and homogenized with a Teflon-glass Potter-Elvehjem ho-
mogenizer. After low speed centrifugation to remove nuclei and unbroken cells, the
resulting supernatant is filtered with gauze and centrifuged twice at high speed. Fi-
nally, the pellets are resuspended in sucrose buffer. The protein content is determined
by the biuret method (Gornall, Bardawill, & David, 1949).

2.3 Materials
Working solutions—all at 0–4 °C
Extraction buffer: 0.25 M sucrose, 0.01 M Tris, 0.1 mM ethylene-bis
(oxoethylenenitrilo) tetraacetic acid, EGTA; pH 7.4 with HCl; 0.4% bovine
serum albumin (BSA)
Sucrose buffer: 0.25 M sucrose, 0.01 M Tris, adjusted to pH 7.4 with MOPS
(3-(N-morpholino)propanesulfonic acid)

2.4 Equipment
Dissecting tools, scissors
Gauze
Potter-Elvehjem homogenizer (clearance 0.1–0.15 mm; 25-mL working volume)
High-speed refrigerated centrifuge with fixed-angle rotor (e.g., RC5B Sorvall
centrifuge, rotor SS34) and suitable polycarbonate tubes
 2 Isolation of mitochondria from rat liver 11

2.5 Protocols
All materials should be pre-cooled and all steps should be performed at 0–4 °C.
1. Starve a 150–175 g male albino Wistar rat overnight and sacrifice by cervical
dislocation. Remove the liver immediately and transfer into a beaker of ice-cold
extraction medium. Wash briefly to remove any remaining traces of blood.
2. Finely mince the liver using scissors.
3. Wash the minced tissue in a beaker containing 50 mL ice-cold extraction buffer.
4. Decant the medium and replace with 40 mL of fresh medium.
5. Homogenize the tissue with a Potter-Elvehjem homogenizer; 8–10 passes with
the homogenizer are required to ensure maximal cell disruption.
6. Dilute the suspension to 60 mL of buffer for one liver and centrifuge at 650  g
for 10 min to remove nuclei and unbroken cells.
7. Carefully filter the supernatant through four layers of gauze and centrifuge it at
17,000  g for 10 min.
8. Discard the supernatant and resuspend the mitochondrial pellet in extraction
buffer without BSA, and centrifuge as above.
9. Discard the supernatant and resuspend the pellet in sucrose buffer at a
concentration 50 mg/mL.

2.6 Safety considerations and standards

All protocols using live animals must conform to governmental regulations regard-
ing the care and use of laboratory animals.

2.7 Analysis and statistics

Typical yields for this procedure are 150–200 mg wet weight of mitochondria per
rat liver. When energized with glutamate/malate, the mitochondria exhibit a
6- to 10-fold increase in oxygen consumption after the addition of ADP, which
indicates that the organelles are largely intact. The respiratory activity assessed
by the P:O ratios (about three for glutamate and two for succinate) approaches
theoretical values.

2.8 Related techniques

RLMs can be the starting material to obtain sub-mitochondrial particles (SMPs)
through sonication. SMPs do not exhibit oxidative phosphorylation activity. How-
ever, since intact mitochondria are not permeable to NADH, SMPs are a useful
model to study NADH-dependent activities. The method used in our experiments
is essentially the one described by Gregg (1967). Mitochondria are prepared from
20 to 30 g of rat liver and the buffer used is the same as that used for the preparation
of broken SMP particles from BHM (Lee & Ernster, 1967), with the addition of 0.4%
BSA from the beginning of the isolation procedure.
12 CHAPTER 1 Isolation of mitochondria from cells and tissues

2.9 Pros and cons

Pros Cons

Ready availability of rat liver to most laboratories Low purity

Mild homogenization conditions compared to other tissues
Rapid preparation
High yield

2.10 Alternative methods/procedures

RLM can be further purified using density gradients. The method described by
Reinhart, Taylor, and Bygrave (1982) allows one to obtain functionally intact
and relatively uncontaminated mitochondria using a discontinuous Percoll gradi-
ent. An alternative method for obtaining highly purified mitochondria uses
semi-automated cell rupture, termed pump-controlled cell rupture system (PCC).
Upon subsequent purification steps, the mitochondrial fraction meets the quality
and purity required for molecular analyses, e.g., proteomic comparisons or deter-
mination of diverse enzymatic activities (Schmitt, Eberhagen, Weber, Aichler, &
Zischka, 2015).

2.11 Troubleshooting and optimization

Problem Solution

Low quality of The animals are normally deprived of food overnight to reduce
mitochondria the glycogen content of the liver; this facilitates the separation
process.
Low respiratory control The clearance between the glass wall and the side of a pestle
must be 0.3–0.5 mm. Use of a pestle that is too tight, or
excessive homogenization, will damage the mitochondria.
High yields and quality also depend on the gentle and rapid
handling of the tissue. For a whole liver, divide the starting
material into 2–3 portions and homogenize them separately.
Removal of lipid is essential, as free fatty acids are potent
uncouplers.
Low purity of isolated Contamination with other organelles can be reduced by
mitochondria washing the mitochondrial pellet gently.
 3 Isolation of mitochondria from beef heart 13

3 Isolation of mitochondria from beef heart

3.1 Definition
The bovine heart is useful for the isolation of mitochondria for two reasons. First,
heart muscle tissue can be obtained in large amounts, which can yield several grams
of mitochondrial proteins from a single isolation procedure. Second, heart muscle
mitochondria do not exhibit the age-associated declines in function observed in
rat liver mitochondria (Azzone, Colonna, & Ziche, 1979). Here, we described a
method, modified from the original paper by Smith (1967), to isolate bovine heart
mitochondria (BHM) that have good enzymatic activity and are stable up to 1 year
when stored at 20 °C.

3.2 Rationale
Differential centrifugation is the most widely used method for obtaining large
amounts of mitochondria for respiratory studies in a relatively short time. The
method is often preferred to gradient centrifugation because it avoids the use of long
density gradients, which can expose the mitochondria to potentially damaging g
forces and unsuitable buffers (Graham, 2001). To isolate BHM, hearts must be re-
moved from animals immediately after slaughter and kept on ice during transport
to the laboratory. The hearts are then cut roughly and ground with a commercial meat
grinder. After this phase, the ground tissue is washed and homogenized with a
blender. Once homogenized, the suspension is centrifuged at low speed to remove
intact cells and nuclei, and then centrifuged again at high speed to obtain a mitochon-
drial pellet. All operations must be carried out in a cold room at 4 °C and the pH con-
stantly adjusted to 7.8 with Tris base solution.

3.3 Materials
One or two beef hearts (approximately 2–2.5 kg) freshly isolated and stored in ice.

3.4 Working solutions

Washing solution: 0.15 M KCl
Sucrose buffer: 0.25 M sucrose, 10 mM Tris, pH 7.6 with HCl
1 M Tris base

3.5 Equipment
Commercial meat grinder
Waring blender
Cheesecloth
Teflon/glass Potter-Elvehjem homogenizer (clearance: 0.1–0.15 mm; volume
30 mL)
14 CHAPTER 1 Isolation of mitochondria from cells and tissues

Low-speed centrifuge with fixed angle rotor or swinging bucket rotor and
250–750 mL bottles
High-speed centrifuge with fixed-angle rotor and 50 mL polycarbonate tubes
Cold room

3.6 Protocol
All subsequent procedures are carried out at 4 °C.

1. Freshly isolated bovine hearts are cleaned of connective and fat tissue and cut into
small cubes.
2. Tissue is washed with washing solution (approximately 2.5 L), passed through a
commercial meat grinder, and resuspended in sucrose buffer (approximately
200 mL of sucrose buffer for 100 g of tissue).
3. The suspension is homogenized for 60 s at high speed in a Waring blender (two
30 s pulses with a 10 s pause between pulses). The pH of the suspension must be
adjusted to 7.5 with 1 M Tris.
4. The homogenate is centrifuged for 10 min at 1000  g to remove unruptured
muscle tissue and nuclei.
5. The supernatant is filtered through two layers of cheesecloth to remove lipid
granules and then centrifuged for 30 min at 26,000  g.
6. The mitochondrial pellet obtained is resuspended in 4 volumes of sucrose
buffer, homogenized with a Teflon/glass Potter-Elvehjem homogenizer (10
strokes using large clearance pestle) and then centrifuged at 26,000  g for
30 min.
7. The pellet is resuspended in the sucrose buffer (approximately 40–50 mL) and
stored at 80 °C, at a protein concentration of 40 mg/mL.

3.7 Analysis and statistics

The average yield of intact BHM is approximately 1 mg protein per gram of start-
ing mince. The protein content is determined by the biuret method (Gornall et al.,
1949). The purity of the preparation is assessed spectrophotometrically by cyto-
chrome content assay (Azzone et al., 1979). Intact beef heart mitochondria should
possess a cytochrome aa3 content of 0.5 μmol/g protein and a cytochrome ratio
c1/c/aa3 of 0.5:1:1. BHM have a P:O ratio in the presence of 300 μM of ADP (state
3 respiration) of about 3 using pyruvate/malate as a substrate and about 2 in the
presence of succinate, with a rate of substrate oxidation of 0.15–0.30 nAtoms
of oxygen/min/mg protein and 0.05–0.09 nAtoms of oxygen/min/mg protein, res-
pectively (Azzone et al., 1979; Smith, 1967). The respiratory control, expressed as
the ratio between state of respiration and state 4 respiration (without ADP), ranges
between 3 and 6 with pyruvate-malate and between 2 and 3 in the presence of
succinate.
 3 Isolation of mitochondria from beef heart 15

3.8 Related techniques

BHM preparation obtained with the method described above can be used for the
preparation of submitochondrial particles (SMP) (Lee & Ernster, 1967) and coupled
submitochondrial particles (phosphorylating electron transport particles, ETPH)
(Beyer, 1967a). SMP are broken membrane fragments with good activity of enzymes
such as NADH-coenzyme Q (CoQ) oxidoreductase and ubiquinol-cytochrome c ox-
idoreductase that are used for the kinetic characterization of Complexes I and III,
respectively (Estornell, Fato, Pallotti, & Lenaz, 1993; Fato et al., 1993). ETPH
are fully capable of undergoing coupled oxidative phosphorylation (Beyer, 1967a;
Hansen & Smith, 1964). Moreover, BHM are a starting material for the preparation
of R4B, a crude mitochondrial fraction enriched in Complex I and Complex III
(Rieske, 1967). The R4B fraction can be further purified to obtain isolated Complex
I, Complex III, Complex IV, and ATPase (Beyer, 1967b; Hatefi, 1978).

3.9 Pros and cons

Pros Cons

High yield Low purity

Inexpensive procedure Need for suitable equipment for large volumes of buffers and
High stability of the a cold room
mitochondria Time consuming procedure
Does not require
ultracentrifugation

3.10 Troubleshooting and optimization

Problem Solution

Low P:O Distilled water used for the isolation media and for the assay reagents must be
ratio of the highest quality.
Resuspend the pellets more gently.
The temperature must be maintained at 4 °C during the entire isolation
procedure.
Low purity The mitochondrial pellet has 2 layers, an upper part (light brown
mitochondria) and a lower part (dark brown mitochondria). Light brown
mitochondria are usually less pure, with lower P:O ratio and with fewer
cytochromes aa3, and can be discarded.
Low yield Homogenization is not sufficient. Use more buffer during the homogenization
and increase the time.
16 CHAPTER 1 Isolation of mitochondria from cells and tissues

4 Isolation of mitochondria from skeletal muscle

4.1 Definition
Biochemical analysis of mitochondria isolated from muscle tissue gives valuable
information on the pathological and physiological characteristics of human diseases
related to impaired mitochondrial metabolism. Mitochondrial bioenergetics can be
carried out both on permeabilized muscle cells or fibers, and on isolated mitochon-
dria. Skeletal muscle mitochondria consist of two separate subpopulations, subsar-
colemmal (SS) and intermyofibrillar (IMF) mitochondria, with different biochemical
properties (Cogswell, Stevens, & Hood, 1993). The optimal isolation buffer for this
preparation—isotonic nonionic solutions containing sucrose vs nearly-isotonic ionic
KCl-containing medium—has been a matter of debate. Overall, the latter isolation
buffer is considered more suitable for isolation of skeletal muscle mitochondria
because skeletal muscle tissue homogenized in isotonic sucrose has a gelatinous con-
sistency (Estabrook, 1967; Pallotti & Lenaz, 2007). Tissue is limited (<1 g whole
tissue) for preparation of mitochondria from human or mouse model skeletal muscle,
which can lead to low recovery of mitochondria (20–30%, compared to up to
40–50%, as reported only by Rasmussen, Andersen, and Rasmussen (1997)).

4.2 Rationale
The majority of methods utilized for the isolation of the entire mitochondrial popula-
tion from skeletal muscle are based on the differential centrifugation technique. Sev-
eral basic methods are available, most of which were reviewed in the previous edition
of this chapter (Pallotti & Lenaz, 2007). Other methods published recently use differ-
ent centrifugation methods (Bharadwaj et al., 2015; Djafarzadeh & Jakob, 2017;
Garcia-Cazarin, Snider, & Andrade, 2011; Lai et al., 2019). The protocol here de-
scribed is suitable for standard biochemical and functional assays. It is optimized
for low amounts of starting material (<1 g muscle) and uses isotonic sucrose under
conditions where the homogenate does not have a gelatinous consistency.

4.3 Materials, equipment and reagents

Isolation Buffer
Mannitol 0.22 M, Sucrose 0.007 M, Tris 2 mM, 1 mM EDTA, 20 mM HEPES,
pH 7.2
0.4% Albumin
Trypsin (or Nagarse) 0.3 mg/mL
Equipment
Potter-Elvehjem homogenizer consisting of a large clearance (0.1–0.15 mm,
volume 10 mL) Teflon pestle driven into a glass vessel mechanically (Braun
S homogenizer)
 4 Isolation of mitochondria from skeletal muscle 17

High speed centrifuge: Beckman J2-21 or Sorvall RC-5B

Fixed angle rotor: Beckman JA-20 or Sorvall SS-34
Centrifuge Tubes: Polycarbonate, 10 mL

4.4 Protocols
All procedures should be performed on ice; all consumables and tubes should be pre-
chilled in ice; centrifuge should be set at 4 °C.

1. 100 mg of muscle tissue (freed from collagen and nerves).

2. Place the tissue in Isolation buffer (approximately 1 mL) containing 0.4%
albumin and mince in small pieces.
3. Incubate the minced tissue in Isolation buffer (+ albumin 0.4%) with trypsin
0.3 mg/mL (100 mg tissue in 0.5 mL) for 5 min at RT, then place immediately
on ice.
4. Dilute in 10 volumes of Isolation buffer (+ albumin 0.4%) without trypsin.
5. Homogenize the tissue (10 strokes using large clearance pestle).
6. Centrifuge at 800  g for 10 min.
7. Collect the supernatant.
8. Resuspend the crude nuclear fraction in the pellet in 2.5 mL of Isolation buffer
with albumin and homogenize the solution (10 strokes using large clearance
pestle).
9. Centrifuge at 800  g for 10 min.
10. Collect the supernatant.
11. Combine the supernatants obtained from steps 7 and 10.
12. Centrifuge at 17,000  g for 10 min.
13. Wash the mitochondrial pellet twice in 2.5 mL of Isolation buffer and centrifuge
at 17,000  g for 10 min.
14. Resuspend the pellet in 50–100 μL of the same buffer.

Expected yield: approximately 0.5 mg of mitochondrial protein from 100 mg of mus-

cle tissue.

4.5 Related techniques

Alternative techniques are optimized for different amounts of starting material or
applications. The method described by Djafarzadeh and Jakob is designed for the
isolation of mitochondria from 5 to 10 g of skeletal muscle (Djafarzadeh & Jakob,
2017). Lai and colleagues published a method for isolation of subsarcolemmal
(SS) and intermyofibrillar (IMF) mitochondria (Lai et al., 2019).
18 CHAPTER 1 Isolation of mitochondria from cells and tissues

4.6 Pros and cons

Pros Cons

Fast method Respiratory experiment should be performed within 1 h

of isolation.
Inexpensive and very efficient Force and speed of the pestle in the homogenization
procedure step can vary between operators.
Mitochondria can be used for
respirometry assays
Minimal quantities of skeletal
muscle required

4.7 Alternative methods/procedures

Mitochondrial respiration in skeletal muscle can be measured in permeabilized mus-
cle fibers (Gnaiger, 2009) by using Oxygraphy for High-Resolution Respirometry
(HRR) or directly on muscle homogenates (Pecinova, Drahota, Nuskova,
Pecina, & Houstek, 2011).

4.8 Troubleshooting and optimization

Problem Solution

Low yield Modify the ratio of tissue:isolation medium (the ideal ratio is 1:5
to 1:10); make sure to perform all the isolation procedures on
ice.
Low quality of isolated Remove the “fluffy” layer (which contains broken mitochondria
mitochondria and fragmented mitochondrial membranes) of the
mitochondrial pellet. Further purify the dark brown pellet
containing intact mitochondria by using a discontinuous
gradient (bearing in mind that some material can be lost by
using this step).

5 Isolation of mitochondria from cultured cells

5.1 Definition
Human cultured cells represent a valid experimental model for investigating mito-
chondrial function, both in physiological and pathological states. In particular, the
transformed cells are suitable for the isolation of mitochondria, as they are easily
cultured and can be obtained in large amounts. Mitochondria can also be isolated
from primary cell cultures (e.g., fibroblasts).
 5 Isolation of mitochondria from cultured cells 19

5.2 Rationale
The method described here is a modification of Yang et al. (1997). The yield varies
with the type of cell line used. For example, yield is typically high from HeLa cells,
which are rich in mitochondria.
Two days before preparation, seed cells in tissue culture dishes. Cultured cells are
detached from dishes by trypsinization and concentrated by centrifugation. The cell
pellet is washed and cells are homogenized using a Potter-Elvehjem homogenizer.
The mitochondria are isolated by differential centrifugation and the protein content
determined by the biuret method (Gornall et al., 1949).

5.3 Materials
Cell pellet derived from tissue culture cells (approximately 5  106–1  107 cells)
Extraction buffer: 0.25 M sucrose, 20 mM HEPES-KOH, pH 7.5, 10 mM KCl,
1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol,
0.1 mM PMSF
Phase contrast microscope
Refrigerated bench top centrifuge
Teflon/glass Potter-Elvehjem homogenizer (clearance 0.1–0.15 mm; volume
5 mL)

5.4 Protocol
Glassware and buffer must be precooled in an ice-bath. Steps 3–9 must be per-
formed at 0–4 °C to minimize the activation of damaging phospholipases and
proteases.

1. Remove the medium from the cells and wash the cells once with PBS.
2. Remove PBS and detach the cells by suitable trypsinization condition.
3. Collect the cells by centrifugation (300  g for 3 min).
4. Wash the cells once with PBS and centrifuge, as above.
5. Discard the supernatant and resuspend with 5 volumes of extraction buffer.
6. The cellular suspension is homogenized with a Teflon-glass homogenizer with
10–20 up-and-down passes of the pestle.
7. The homogenate is then centrifuged at 750  g for 10 min.
8. The resulting supernatant is transferred to a pre-chilled centrifuge tube and stored
on ice, and the pellet is resuspended in extraction buffer, homogenized and
centrifuged as for steps 5–7.
9. The supernatants obtained from the two low-speed spins are pooled and then
centrifuged at 10,000  g for 15 min. Crude mitochondria, which are
recovered in the pellet, are resuspended in extraction buffer (approximately
20–25 μL).
20 CHAPTER 1 Isolation of mitochondria from cells and tissues

5.5 Analysis and statistics

The crude mitochondria isolated using this method can be used for polarographic
determinations, spectrophotometric determinations, and protein analysis, such as
Western blots and ELISA. The yield depends on the type of cells and can vary from
7 to 50 μg of mitochondrial protein per 106 cells. For experiments requiring a high
degree of purity (e.g., in vitro protein synthesis and proteomic studies), separation
methods such as gradient centrifugation must be used (see related techniques sec-
tion). The purity and the enrichment of mitochondria from whole cells can be
assessed by Western blot analyses, measuring cytosolic (e.g., actin), nuclear (e.g.,
lamins A/C), and mitochondrial (e.g., OXPHOS subunits and porin) marker proteins.
The integrity of the mitochondrial fraction can be tested by quantitation of an inter-
membrane space protein (e.g., cytochrome c) or a matrix protein (e.g., cyclophilin D)
by immunoblot or by measuring ADP stimulation by polarography. Alternatively,
measuring mitochondrial Ca2+ buffering capacity is a fast and efficient method to
assess the integrity of isolated mitochondria (Wettmarshausen & Perocchi, 2017).

5.6 Related techniques

Mitochondria of higher purity can be prepared using a sucrose gradient. A valid
method, which is a modification of the “two-step” procedure described by Tapper,
Van Etten, and Clayton (1983), has been described by Magalhaes, Andreu, and
Schon (1998). An alternative method to mechanical homogenization of cells is nitro-
gen cavitation (Gottlieb & Adachi, 2000; Simpson, 2010). Mitochondria prepared by
this method possess an intact outer membrane, high respiratory control ratios, and re-
tain intermembrane space components, including cytochrome c and adenylate kinase.

5.7 Pros and cons

Pros Cons

Rapid method: the entire protocol The integrity of mitochondria must be checked
takes about 90 min carefully after isolation.
Cultured cells are standardized All the experiments that require intact mitochondria
models and easily obtained must be performed within 3 h after isolation.
The protocol for mitochondria isolation must be
adapted depending on cell type.

5.8 Alternative methods/procedures

Mitochondria can be isolated from mammalian cells or tissues through the use of
magnetic beads that are covalently coupled to antibodies that recognize mitochon-
drial outer membrane proteins (e.g., Tom20) (Hornig-Do et al., 2009). However, this
method may alter mitochondrial ultrastructure or cover epitopes in the outer mem-
brane (see Section 6 in this chapter).
6 Affinity purification of mitochondria from yeast using magnetic beads 21

5.9 Troubleshooting and optimization

Problem Solution

Low yield Homogenization has a large impact on yield of the mitochondrial preparation.
We recommend use of a Teflon-glass homogenizer. Homogenization using a
glass-glass homogenizer can lead to increased yield but can also damage
mitochondria.
Visualization of cell disruption using a microscope can be used to optimize
homogenization conditions for different cell lines. The homogenization step
should lyse about 80% of the cells.
Fibroblast cell membranes are difficult to break open. To promote
homogenization, freeze-thaw cells once harvested.
Low Homogenization and all the subsequent steps of the protocol must be
quality performed at 4 °C to minimize the activation of phospholipases and proteases.
Excessive homogenization can cause damage to the mitochondrial membrane
and trigger release of mitochondrial components.
Avoid diluting the mitochondria with buffer. Mitochondria retain their function
for a longer period, probably as a result of less exposure to oxygen, when
stored in a concentrated form (30–50 mg/mL).

6 Affinity purification of mitochondria from yeast using

magnetic beads
6.1 Definition
Crude mitochondria isolated from budding yeast by differential centrifugation
contain other membrane or organelle contaminants (Daum, B€ohni, & Schatz,
1982). While crude mitochondria can be purified further by density gradient ul-
tracentrifugation (Boldogh & Pon, 2007; Glick & Pon, 1995), these methods
require large amounts of starting material and access to an ultracentrifuge. Here,
we describe a method to isolate highly purified mitochondria from yeast using
magnetic bead affinity purification (Liao, Boldogh, Siegmund, Freyberg, & Pon,
2018). This method includes the removal of magnetic beads from mitochondria,
therefore minimizing their interference in subsequent structural or functional
studies.

6.2 Rationale
We tagged the mitochondrial outer membrane protein Tom70 at its chromosomal
locus with 6xHis. Yeast expressing Tom70-6xHis are grown to mid-log phase in
liquid medium containing a non-fermentable carbon source (lactate), which pro-
motes mitochondrial biogenesis. Cells expressing Tom70-6xHis are then treated
with zymolyase, which catalyzes breakdown of the cell wall, and the resulting
22 CHAPTER 1 Isolation of mitochondria from cells and tissues

spheroplasts are disrupted by Dounce homogenization. The cell lysates are sub-
jected to low speed centrifugation to sediment intact cells, nuclei, and cellular
debris, and the resulting supernatant—the mitochondria-enriched fraction—is in-
cubated with Ni-NTA magnetic beads. Mitochondria that are bound to the beads
are separated under magnetic fields, washed, and released from magnetic beads
with imidazole treatment (Fig. 1A).

FIG. 1
Isolation of mitochondria using Ni-NTA magnetic beads. (A) Scheme of mitochondrial
isolation using magnetic beads. Detailed steps are described in the main text. (B) Marker
proteins for mitochondria (Tom70, Porin, cytochrome b2 [Cyb2], α-ketoglutarate
dehydrogenase [Kgd1]), cytosol (hexokinase [Hxk]), ER (Sec61), and vacuole (Nyv1) are
probed using Western blot analysis. Total protein load was assessed using 2,2,2-
trichloroethanol (TCE). (C) Crude or bead-purified mitochondria are treated with (+) or without
( ) 100 μg/mL proteinase K (PK) for 30 min at 4 °C. Mitochondrial outer membrane proteins
(Tom70 and Porin) and an intermembrane space protein (Cyb2) are probed using Western
blot analysis.
6 Affinity purification of mitochondria from yeast using magnetic beads 23

6.3 Materials, equipment and reagents

Strains
PLY31: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 TOM70-6xHis-KanMX6

6.4 Materials
Zymolyase 20T (Seikagaku Corporation, Tokyo, Japan)
HisPur™ Ni-NTA Magnetic Beads (88831, Thermo Scientific, Grand Island, NY)
Stock solutions
1 M Tris–SO4, pH 9.4. Autoclave.
1 M DTT. Dissolve in water. Store at 20 °C.
1 M MgCl2. Autoclave.
2.4 M Sorbitol. Autoclave.
200 mM Phenylmethylsulfonylfluoride (PMSF) (Roche, Indianapolis, IN).
Dissolve in 100% ethanol. Store at 20 °C.
1 M HEPES–KOH, pH 7.4. Adjust pH with KOH. Autoclave.
1 M KPi, pH 7.4. (19.8 mL 1 M KH2PO4 and 80.2 mL 1 M K2HPO4)
Autoclave.
Protease Inhibitor (PI) cocktails (all reagents are from Sigma, St. Louis, MO).
PI-1: 1000  stock dissolved in H2O (Sigma catalog numbers in parentheses):
0.5 mg/mL Pepstatin A in dimethyl sulfoxide (DMSO) (P4265), 0.5 mg/mL
Chymostatin in DMSO (C7268), 0.5 mg/mL Antipain (A6191), 0.5 mg/mL
Leupeptin (L2884), 0.5 mg/mL Aprotinin (A1153).
PI-2: 1000  stock dissolved in 100% ethanol: 10 mM Benzamidine–HCl
(B6506), 1 mg/mL 1,10-Phenanthroline (P9375).
Both cocktails are divided into 0.5 mL aliquots and stored at 20 °C.

Working solutions
Lactate medium: 3 g/L Yeast extract (Difco), 0.5 g/L Glucose, 0.5 g/L
CaCl2
2H2O, 0.5 g/L NaCl, 0.6 g/L MgCl2
6H2O, 1 g/L KH2PO4, 1 g/L
NH4Cl, 22 mL/L of 90% Lactic acid, 7.5 g/L NaOH, adjust pH to 5.5 with
NaOH pellets.
Tris–DTT buffer: 0.1 M Tris–SO4, pH 9.4, 10 mM DTT.
SP buffer: 1.2 M Sorbitol, 20 mM KPi, pH 7.4.
SEH buffer (add PI-1, PI-2, and PMSF immediately before use): 0.6 M
Sorbitol, 20 mM HEPES–KOH, pH 7.4, 2 mM MgCl2, 1  PI-1, 1 PI-2,
1 mM PMSF.

6.5 Equipment
Sorvall RC5C Refrigerated Centrifuge with Sorvall GS-3 rotor and Sorvall SS34
rotor
New Brunswick Innova 4330 Refrigerated Incubator Shaker
24 CHAPTER 1 Isolation of mitochondria from cells and tissues

40-mL glass/glass Dounce homogenizer (Wheaton Science Products, Millville, NJ)

6-Tube Magnetic Separation Rack (New England Biolabs, Ipswich, MA)
Multipurpose tube rotator (Fischer Scientific, Hampton, NH)

6.6 Protocols
1. Prepare precultures by inoculating a colony of yeast expressing 6xHis-tagged
Tom70 into 5 mL of lactate medium in a 50-mL Falcon tube and incubate at 30 °C
with aeration (200rpm in a New Brunswick shaking incubator) overnight. For
1.5 L growths prepare 3 precultures.
2. Inoculate 1.5 L lactate medium in a 4.5 L shake flask with the 15 mL preculture.
Grow yeast cells to mid-log phase at 30 °C overnight with aeration, as above.
3. Concentrate cells by centrifugation at 1500  g for 5 min at 4 °C (3500 rpm in a
Sorvall GS-3 rotor).
4. Discard the supernatant and wash cells by resuspending the cell pellet with
200 mL water and concentrating them by another round of centrifugation at
1500  g for 5 min at 4 °C. Remove the supernatant and weigh the “wet” cell
pellet. Typically, we obtain about 4 g wet cells from a 1.5 L culture.
5. Resuspend the cell pellet in 20 mL of Tris–DTT buffer in a 125 mL Erlenmeyer
flask, and incubate for 15 min at 30 °C with shaking (200 rpm in a New
Brunswick shaking incubator). Transfer the suspension to Sorvall SS34 rotor
tubes, and centrifuge at 1500  g for 5 min at 4 °C.
6. Resuspend the cell pellet in 20 mL of SP buffer, and centrifuge at 1500  g for
5 min at 4 °C. To convert yeasts to spheroplasts (cells without cell walls),
resuspend cells in 20 mL of SP buffer containing Zymolyase 20T (7.5 mg/g
yeast wet) and incubate in a 125-mL Erlenmeyer flask at 30 °C for 40 min with
shaking (200 rpm in a New Brunswick shaking incubator).
7. Transfer the spheroplast suspension to a SS35 Sorvall tube and centrifuge at
4500  g (6000 rpm in a Sorvall SS34 rotor) for 5 min at 4 °C. Resuspend the
pellet in 20 mL of ice-cold SEH buffer. Keep everything on ice in the following
steps.
8. Centrifuge the mixture at 4500  g for 5 min at 4 °C. Resuspend the pellet of
spheroplasts in 20 mL of ice-cold SEH buffer, and transfer to a prechilled 40-mL
glass/glass Dounce homogenizer. Using the tight-fitting pestle, homogenize the
spheroplasts with 15 forceful strokes.
9. Centrifuge the homogenate at 1500  g for 5 min at 4 °C. Collect the
supernatant and centrifuge at 12,000  g for 10 min at 4 °C. Resuspend the
pellets in 4 mL ice-cold SEH buffer. We refer to this fraction as the
“mitochondria-enriched fraction.”
10. Prepare HisPur™ Ni-NTA Magnetic Beads, as follows. Transfer 100 μL beads
to a 1.5-mL microcentrifuge tube (100 μL beads/1 mL mitochondria-enriched
fraction). Place the tube in a Magnetic Separation Rack for 30 s to concentrate
6 Affinity purification of mitochondria from yeast using magnetic beads 25

beads at the lateral surface of the tube adjacent to the magnet. Remove the
residual liquid using a micropipette. Remove the tube from the separation rack,
and wash beads with 500 μL SEH buffer 2 times.
11. Incubate 1 mL of mitochondria-enriched fraction with washed beads for
10–60 min at 4 °C with rotation (low speed) in a multipurpose tube rotator.
Place the mixture in the separation rack for 1 min to separate the magnetic bead-
bound mitochondria from other organelles or debris. Wash the bead-bound
mitochondria 3 times with 15 mM imidazole in ice-cold SEH buffer.
12. To elute mitochondria, incubate magnetic bead-bound mitochondria with 50 μL
of 500 mM imidazole in ice-cold SEH buffer for 5 min with rotation. Place the
tube in the magnetic field to trap the beads. Transfer the released mitochondria
to a microcentrifuge tube and centrifuge at 12,000  g for 5 min at 4 °C.
Remove the supernatant and resuspend the mitochondrial pellet in ice cold SEH
buffer, as needed.

6.7 Analysis and statistics

6.7.1 Examination of mitochondrial purity
We typically obtain about 100 μg of mitochondria from 1 g of wet yeast after incu-
bation of the mitochondria-enriched fraction with the magnetic beads for 60 min. The
yield obtained after 10-min incubation is reduced by 25% compared to that obtained
after a 60-min incubation.
To compare the purity of mitochondria preparations using magnetic beads and
differential centrifugation, we perform Western blot analysis using antibodies
against marker proteins of the mitochondrial outer membrane (Tom70 and porin),
intermembrane space (cytochrome b2, Cyb2), matrix (α-ketoglutarate dehydroge-
nase, Kgd1), ER (Sec61), cytosol (hexokinase), and vacuole (the vSNARE Nyv1).
Mitochondrial proteins Tom70, porin, Cyb2, and Kgd1 are expected to enrich in
crude and magnetic bead-purified mitochondria. In addition, cytosolic, ER, and
vacuole contamination in bead-purified mitochondria are expected to be lower com-
pared to that observed in crude mitochondria (Fig. 1B). These indicate that mito-
chondria isolated using the magnetic bead method are purer compared to crude
mitochondria prepared by differential centrifugation.

6.8 Characterization of mitochondrial integrity

To characterize the integrity of mitochondria, we treat them with proteinase K. If
mitochondria are intact, outer membrane proteins (Tom70) are expected to be
protease-sensitive, and the intermembrane space protein (cytochrome b2) is expected
to be protease-protected. Porin, an outer membrane protein that is protease-resistant is
used as a protein loading control (Fig. 1C).
26 CHAPTER 1 Isolation of mitochondria from cells and tissues

6.9 Related techniques

Other magnetic bead-based isolation methods are included as follows. Magnetic beads
coated with antibodies have been used to isolate mitochondria in mammalian systems
from cells (Hornig-Do et al., 2009), mouse tissues (Franko et al., 2013), and human
cortex (Khattar et al., 2016). Lipophilic and delocalized triphenyl phosphonium
(TPP) cation-bound magnetic beads, which recognize mitochondria by mitochondrial
membrane potential (Δψ), have also been applied to isolate mitochondria (Banik,
Askins, & Dhar, 2016; Banik & Dhar, 2017). However, mitochondria isolated using
these methods cannot be released from magnetic beads, which may affect mitochon-
drial ultrastructure and/or cover epitopes on the mitochondrial outer membrane.

6.10 Pros and cons

Pros Cons

Generates highly pure mitochondria. Not suited for large amounts of

mitochondrial preparation.
Less time-intensive compared to other methods for
preparation of highly purified mitochondria.
Mitochondria are released from magnetic beads.
Does not require large amount of starting materials.
Does not require ultracentrifugation.
More cost-effective than commercially-available
magnetic bead purification kits.

6.11 Troubleshooting and optimization

Problem Solution

Low yield Use lactate medium. Other yeast growth medium may lower the
yield.
Yeasts do not convert to spheroplasts. Conditions for spheroplast
formation vary according to the batch of zymolyase and/or the
strains used.
Insufficient homogenization. Use the adequate volume of
homogenizer or increase the number of strokes.
Binding capacity of magnetic beads may vary according to the
batch. Adjust the ratio of beads and mitochondria-enriched fraction.
Mitochondria are not completely eluted. Flip tubes several times
every minute.
Mitochondria are Wash bead-bound mitochondria with a higher concentration of
not pure imidazole in SEH buffer or wash > 3 times. However, high
concentration of imidazole may decrease the yield.
Mitochondria are Reduce the number of strokes in the homogenization steps.
not intact Resuspend pellets more gently.
 References 27

Acknowledgments
This work was supported by grants from the National Institutes of Health (NIH) (GM45735,
GM122589 and AG051047) to L.A.P. C.B, R.F., and F.P. express their gratitude to their men-
tor Prof. Giorgio Lenaz for having introduced them to the exciting field of mitochondrial re-
search, for his guidance, and for his helpful suggestions.

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