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PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF
MITOCHONDRIAL COMPLEX III
MS301 COMPLEX III IMMUNOCAPTURE KIT
Isolates Complex III from human, mouse, rat and bovine tissues and cell lines
immunoprecipitates obtained. The subunits of the complex
are clearly resolved.
RESEARCH USES
The Complex III immunocapture kit allows isolation of the
ubiquinol-cytochrome c reductase complex (E.C. 1.10.2.2)
from small amounts of tissue. This facilitates subsequent
analysis of assembly state, activity and the extent of post
translational modifications including oxidative damage that
occur with aging. Uses for the Complex III immunocapture
kit include research on genetic mitochondrial disease (1),
where complex III may have an additional role in the
stabilization of complex I (2).
DESCRIPTION
The key component of the Complex III immunocapture kit is a
monoclonal antibody able to selectively immunocapture the
enzyme complex. The mAb is already covalently cross linked
to Protein G-Agarose for convenience of use. This material is
provided in batches of 25, 50 and 75 l beads which have
been charged with approximately 250, 500 and 750 g of
antibody respectively. When used as described in the protocol
in Figure 1, 10 l of beads are able to immunocapture
approximately 10 g of Complex III from heart mitochondria.
Also provided are 2 mg of bovine heart mitochondria as a
positive control to be used prior to, or during, isolation of
Complex III from experimental samples. As an alternative,
researchers can purchase the individual components i.e. 100
g of mAb and 2 mg BHM (kit MS301c).
SUGGESTED PROTOCOL FOR IMMUNOCAPTURING
COMPLEX III
The amount of Complex III that is captured in any experiment
depends on both the amount of capture antibody and the
amount of cell extract or isolated mitochondria used.
Calculation of the amount of beads to be used in any
experiment must also take account of the source of the
material from which Complex III is to be isolated because
mitochondria from different tissues have different
concentrations of the enzyme complex. For example the
levels of Complex III in mitochondria from cell culture
material are around 10 fold less that in heart mitochondria.
Figure 1 shows a schematic of a generic protocol developed
for isolating Complex III from heart tissue.
1mg mitochondria in 0.18 ml Buffer A,
1 g/ml pepstatin, 1g/ml leupeptin, 1mM PMSF

Add 20 l 10% LM incubate on ice 30 mins

Centrifuge 72000g 4oC 10 mins.

Incubate supernatant with 10 l capture resin MS301
3 hours at room temperature OR overnight 4oC

Beads collected by gentle centrifugation (10 sec 500g) and
washed for 5 mins in PBS, 0.05 % LM, repeat twice

Complex III eluted from the resin by addition of
50 l 0.1 M glycine, pH 2.5, 0.05 % LM
or 50 l 1 % SDS

Protein concentration determined.
(e.g. by micro-BCA method (Pierce))

Buffer A : 50 mM Tris.HCl pH 7.5
Leupeptin : 1 mg/ml (water)
Pepstatin : 1 mg/ml (ethanol)
PMSF : 0.3 M (ethanol)
LM : n-dodecyl-b-D-maltoside
Figure 1. Example of Complex III isolation
procedure from heart mitochondria
Figure 2 shows the subunit structure of Complex III
immunocaptured from beef heart and human heart
mitochondria by Coomassie blue stained SDS-PAGE of
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PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF MITOCHONDRIAL COMPLEX IV
MATERIALS AND STORAGE
Kit MS301 contains the anti-Complex III immunocapture
mAb covalently linked to protein G-agarose beads in 25, 50 or
75 l amounts. Beads have 8-10 g mAb bound per l bead
volume. All volumes of bead resin are suspended in 400 l of
PBS buffer (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM
NaCl, 2.7 mM KCl, pH 7.3) with 0.02 % sodium azide. Also
included are 2 mg of purified bovine heart mitochondria
resuspended in 400 l of heart mitochondria resuspension
buffer (10 mM Tris.HCl, pH 7.8, 0.2 M sucrose, 0.2 mM
EDTA, 1 mM PMSF). The antibody is shipped on ice. Upon
receipt store the mAb at 4oC. The mitochondrial preparation
should be aliquoted and stored at -20oC until use.
1000 g
BHM
500 g 500 g
BHM HHM
188
98
ADDITIONAL PRODUCTS THAT COMPLEMENT THE
COMPLEX III IMMUNOCAPTURE KIT
MS302 COMPLEX III SUBUNIT WESTERN
BLOTTING KIT
Confirms Complex III enzyme purity
RESEARCH USES
The Complex III Western blotting kit contains three different
mAbs reacting specifically with the core protein 1, the Rieske
protein, and a 10 kDa Complex III subunit of complex III
respectively, along with a mAb against the  subunit of
Complex V (ATP synthase F1) included as a control. This
mAb mixture can be used to probe the isolated complex III for
integrity and purity as shown in Figure 1. Alternatively the
cocktail can be used to conveniently detect the presence of 3
key Complex III proteins relative to a control (Complex V )
in patient samples. Also included in this kit are SDSdissociated human heart mitochondria to use as a control for
the identification of Complex III subunits on the gel system
chosen for analysis.
62
Acyl CoA
deHase
Core 1
Core 2
49
38
ANT
Porin
3
4 c1
5 Rieske
28
17
14
6
7
8
9, 10
6
Figure 2. Immunocapture of Complex III from
human and bovine mitochondria. Using 10 l of
capture resin MS301, the yield of Complex III from
1000 g of bovine heart mitochondria (BHM) is
approximately 10 g. However a yield detectable by
Coomassie staining can be obtained from as little as
250 g of total mitochondria. This yield is enough
for most mass spectrometry applications
Figure 3. Complex III subunit identification. MAb
cocktail M302 can be used to verify the presence of
the core protein 1, Rieske protein and a 10 kDa
subunit in immunocaptured Complex III while using
the ATP synthase F1subunit mAb as a control. The
 subunit is an abundant mitochondrial protein.
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PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF MITOCHONDRIAL COMPLEX IV
MATERIALS AND STORAGE
Kit MS302 contains a cocktail of antibodies totaling 200 g
(MS302-200) or 400 g (MS302-400). The mixture is
comprised of 4 mAbs reactive to Complex III Core protein 1
(50 kDa), the Rieske protein (25 kDa) and a 10 kDa subunit of
complex III and Complex V in PBS buffer (1.4 mM
KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, pH
7.3), supplemented with 0.02 % sodium azide. The mAb
cocktail is shipped on ice; upon receipt store at 4oC. Also
supplied are human heart mitochondria solubilized in SDSPAGE loading buffer (2 % SDS, 60 mM Tris.HCl, pH 6.8, 5
% glycerol, 0.02 mg/ml bromophenol blue). This control
mitochondrial sample should be stored at 4oC or for longer
periods of time at -20oC.
ANTI-COMPLEX III SUBUNIT ANTIBODIES FOR
VARIED APPLICATIONS
Monoclonal antibodies are available to four Complex III
subunits for varied applications such as Western blotting
Immunocytochemistry, ELISA of human samples (see Figure
4).
MS303 anti-Complex III Core Protein 1 mAb for Western
blotting
MS304 anti-Complex III Core Protein 2 mAb for Western
blotting and Immunocytochemistry (see Figure 5).
MS305 anti-Complex III FeS (Rieske protein) mAb for
Western blotting
MS306 anti-Complex III-10 kD mAb for Western blotting.
Figure 5. Immunocytochemical labeling of human
MRC5 fibroblast mitochondria with MS304 an anti
Complex III core 2 antibody. In this example the
anti-Core 2 mouse monoclonal antibody is
visualized by the addition of an Alexa 488 Goat anti
mouse antibody (Molecular Probes Inc).
MATERIALS AND STORAGE
These products contain 100 g of mAb (MS303-306)
dissolved in 100 l of PBS, 0.02% azide. The mAb is shipped
on ice; upon receipt store at 4oC. Also supplied as a Western
blot control sample are human heart mitochondria solubilized
in SDS-PAGE loading buffer (2 % SDS, 60 mM Tris.HCl, pH
6.8, 5 % glycerol, 0.02 mg/ml bromophenol blue). This
control mitochondrial sample should be stored at 4oC or for
longer periods of time at -20oC.
(MS601) WESTERN BLOTTING DETECTION KIT FOR
OXPHOS COMPLEXES
A mAb cocktail optimized for the detection of a subunit from each of the 5
OXPHOS complexes
RESEARCH USES
Figure 4. Western blotting using complex III
monoclonal antibodies.
This kit is an already mixed solution containing 5 mAbs, one
each against a 20 kDa subunit of Complex I, 30 kDa nonheme iron subunit of succinate dehydrogenase (Complex II),
core protein 2 of Complex III, subunit II of cytochrome c
oxidase (Complex IV) and the  subunit of the ATP synthase
(Complex V). These polypeptides are well separated in SDS-
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PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF MITOCHONDRIAL COMPLEX IV
Cocktail
V
F1
III
II
IV
core 2 Ip 30kDa COXII
I
20kDa
PAGE as shown in Figure 6. The OXPHOS mAb cocktail
provides an important analysis of the purity of OXPHOS
complexes after immunocapture. However this cocktail could
also be used to detect a specific reduction in the levels of an
individual OXPHOS complex with respect to the others. Also
included are SDS-dissociated human heart mitochondria as an
SDS-PAGE control.
MATERIALS AND STORAGE
Figure 6. Simultaneous blotting of a representative
subunit from all 5 OXPHOS complexes. In this figure
each lane contains 10 g of MRC5 fibroblast
mitochondria. mAb cocktail M601 is shown in the
first lane and for demonstrative purposes the
individual mAbs making up this cocktail are shown
alongside, these are a 20 kDa subunit (Complex I), 30
kDa Ip subunit (Complex II), core 2 (Complex III),
COX II (Complex IV) and F1 (ATP synthase)
respectively.
Kit MS601 is available in 300 g (MS601-300) or 600 g
(MS601-600) amounts in PBS (0.02% azide) of each of 5
mAbs against I-20 kDa, II-30 kDa, III-core2, IV-COXII and
ATP synthase F1. The mAb cocktail is shipped on ice; upon
receipt store at 4oC.
Also supplied are human heart
mitochondria solubilized in SDS-PAGE loading buffer (2 %
SDS, 60 mM Tris.HCl, pH 6.8, 5 % glycerol, 0.02 mg/ml
bromophenol blue). This control mitochondrial sample should
be stored at 4oC or for longer periods of time at -20oC.
1.
2.
J Clin Invest (2003) 111, 303-312
Mol Cell (2004) 13, 805-815
Price List
 MS301-250 Kit to immunocapture ~25 g of Complex III from heart mitochondria plus 2 mg of BHM control. . . . . . . . . . . . . . . . . . . . . . . . $295
 MS301c contains 100 g of Complex III immunocapture antibody plus 2 mg of BHM control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. $270
 MS301-500 Kit to immunocapture ~50 g of Complex III from heart mitochondria plus 2 mg of BHM control. . . . . . . . . . . . . . . . . . . . . . . . .$550
 MS301-750 Kit to immunocapture ~75 g of Complex III from heart mitochondria plus 2 mg of BHM control. . . . . . . . . . . . . . . . . . . . . . . . .$795
 MS302-200 Complex-III Subunit Western Blotting Kit containing a mixture of mAbs totaling 200 g plus HHM control . . . . . . . . . . . . . . . . $375
 MS302-400 Complex-III Subunit Western Blotting Kit containing a mixture of mAbs totaling 400 g plus HHM control . . . . . . . . . . . . . . . . $695
 MS303 Western Blotting Detection of Core-1 (100 g mAb) plus HHM control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . $250
 MS304 Western Blotting Detection of Core-2 (100 g mAb) plus HHM control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . $250
 MS305 Western Blotting Detection of Rieske FeS protein (100 g mAb) plus HHM control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .$250
 MS306 Western Blotting Detection of a Complex III 10 kDa subunit (100 g mAb) plus HHM control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . $250
 MS601-300 Total OXPHOS Complexes Western Blotting Detection Kit containing a mixture of mAbs totaling 300 g plus HHM control. . . $375
 MS601-600 Total OXPHOS Complexes Western Blotting Detection Kit containing a mixture of mAbs totaling 600 g plus HHM control. . . $695

All MitoSciences products are sold “FOR RESEARCH PURPOSES ONLY”
MitoScience LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801
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