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PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF
MITOCHONDRIAL OXPHOS COMPLEXES
ANTIBODIES FOR USE IN THE DETECTION OF
OXPHOS COMPLEXES AFTER BLUE NATIVE
ELECTROPHORESIS
Individual mAbs for first dimension BN-PAGE OXPHOS protein detection
and a mAb cocktail MS603 for second dimension BN-PAGE OXPHOS
protein detection.
RESEARCH USES
Blue Native electrophoresis is a simple and effective way to
sub-fractionate multisubunit complexes (1). This technique
allows the determination of the assembly states of all
mitochondrial multisubunit OXPHOS complexes. Altered
levels of assembly can arise from mutations in structural
proteins, mutations in complex-specific assembly factors (2,3),
or mtDNA depletion (4). This kind of analysis has been
performed with tissue samples, platelets and cultured
fibroblasts from human subjects and patients (5,6). However,
in many cases the quantities of these samples are small and
their mitochondria content are low.
For this reason
MitoSciences provides monoclonal antibodies optimized for
the rapid and sensitive detection of OXPHOS proteins and
enzyme complexes separated by Blue Native electrophoresis.
Figure 1 shows a schematic protocol for the separation of
mitochondrial proteins by Blue Native polyacrylamide gel
electrophoresis (BN-PAGE) however a more complete
description of BN-PAGE sample preparation and analysis can
found at www.mitosciences.com. In BN-PAGE multisubunit
enzymes bind a charged coomassie dye which allows their
electrophoretic separation by enzyme size in the 1st dimension.
In an optional second electrophoretic step can be employed
whereby the proteins within each complex are further resolved
by SDS-PAGE in the perpendicular direction. MitoSciences
makes available individual monoclonal antibodies which can
detect OXPHOS complexes after the first dimension
separation (see below) and also a cocktail of 5 monoclonal
antibodies, MS603, for the rapid and highly sensitive detection
of OXPHOS proteins after this second dimension. This
cocktail offers increased sensitivity and opportunity for the
simultaneous analysis of the assembly state of all 5 OXPHOS
complexes from a single patient sample.
400 g mitochondria in 40 l Buffer A,
1 g/ml pepstatin, 1g/ml leupeptin, 1mM PMSF

Add 7.5 l 10% LM incubate on ice 30 min

Centrifuge 72000g 4oC 10 min

Add 2.5 ml of a 5 % suspension of
Coomassie blue G in buffer A

Load samples on 6-13 % Native acrylamide gradient gel.
Gel recipe and electrophoresis buffers described in BN-PAGE
manual.

1st dimension - For single dimension analysis; proteins should be
electroblotted for antibody detection according to standard
protocols.

2nd dimension - For two dimension analysis; entire gel lane
should soaked in SDSPAGE sample buffer, then loaded onto
SDS-denaturing gel.

Buffer A : 0.75 M 6-aminocaproic acid,
50 mM Bis-Tris/HCl pH 7.0
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 Blue Native procedure for
separation of mitochondrial proteins according to
Schägger and von Jagow (1)
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DESCRIPTION OF BN-PAGE FIRST DIMENSION MABS
Each of the 5 OXPHOS complexes can be detected after 1st
dimension BN-PAGE by immunoblotting with a mAb from
MitoSciences. These same antibody clones have been used
for the analysis of patient fibroblast after 1st dimension BNPAGE in recent work by Ugalde et al. (7).
MS105
MS111
MS103
Complex I ND6
Complex I NDUFA9 subunit
Complex I Grmi-19
MS204
MS304
MS404
MS507
Complex II 70 kDa subunit
Complex III core 2 subunit
Complex IV COXI subunit
Complex V subunit
receipt store at 4oC. 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). This control
mitochondrial sample can be used as a blotting control or
even a Blue Native separation control and should be stored
at -20oC until use. The mAb cocktail Kit MS-603 is
available in 300 l (MS-603-300) or 600 l (MS-603-600)
amounts in PBS (0.02% azide). This is a premixed cocktail
of 5 mAbs against I-NDUFA9, II-70 kDa, III-core2, IVCOXIV and ATP synthase F1.
With 3 mAbs available which are reactive against complex I
i.e. anti NDUFA9, anti Grim19 (MS103) and anti 20 kDa
subunit (MS105) this allows greater definition of any
Complex I subassemblies formed. When these antibodies are
purchased for this BN-PAGE blotting application a sample of
bovine heart mitochondria can be supplied upon request which
will act as a good control for optimal resolution of the
OXPHOS complexes on the gel system chosen.
DESCRIPTION OF BN-PAGE mAb COCKTAIL MS603
FOR SECOND DIMENSION ANALYSIS
The Blue Native PAGE analysis of OXPHOS enzymes from
fibroblasts or blood samples is made more difficult due to a
limiting amount of protein. The Blue Native OXPHOS
Western blotting kit MS603 is a cocktail of mAbs which
allows the rapid and highly sensitive detection of these
OXPHOS components from very small sample amounts.
This cocktail allows the simultaneous analysis of the
assembly state of all 5 OXPHOS complexes from a single
patient sample as shown in Figure 2. Each subunit
identified by kit MS603 has been shown in studies of
patients with OXPHOS deficiencies to be stable when that
complex fails to assemble properly i.e. it may be retained in
a partial assembly. Therefore the kit can be used to study
the state of assembly in samples where mitochondrial
dysfunction is suspected. Note that the mAbs in this kit are
different from those in kit MS601 (which includes subunits
readily lost and proteolytically turned over when full
assembly is not achieved).
MATERIALS AND STORAGE
The monoclonal antibodies shown in MS103, 105, 111, 204,
304, 404, and 502 contain 100 g of mAb dissolved in 100
l of PBS, 0.02% azide. The mAb is shipped on ice; upon
Figure 2. Two dimension Blue Native PAGE analysis
of fibroblasts that are (A) normal and (B) complex I
deficient. It is clear that the complex I deficient cell line
shown in B shows no detectable level of complex I,
however all other OXPHOS complexes appear normal
including a minor amount of heterocomplex formed
between complexes III and IV which is a well
documented species (8). Only a minor mitochondrial
enrichment was performed upon these samples cell
lines. Each sample represents only 8% of confluent cells
taken from a 10 cm diameter tissue culture dish.
MitoSciences LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801
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The mAb cocktail is shipped on ice; upon receipt store at 4 oC.
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). This control mitochondrial sample can
be used as a blotting control or even a Blue Native separation
control and should be stored at -20oC until use.
1.
2.
3.
4.
5.
6.
7.
8.
Anal Biochem (1991) 199, 223-231
Curr Med Chem (2004) 11, 233-239
Embo J (2002) 21, 43-52
J Clin Invest (2003) 111, 303-312
Anal Biochem (1995) 231, 218-224
Electrophoresis (1996) 17, 709-714
Hum Mol Genet (2004) 13, 659-667
J Biol Chem (2000) 275, 18093-18098
Price List
 MS603-300 Total OXPHOS Complexes Blue Native Blotting Detection Kit containing a mixture of mAbs totaling 300 g . . . . . . . . .. . . . . . . . . . . . . .$375
 MS603-600 Total OXPHOS Complexes Blue Native Blotting Detection Kit containing a mixture of mAbs totaling 600 g . . . . . . . . .. . . . . . . . . . . . . .$695
 MS103 Blue Native, Western Blotting and Immunocytochemistry Detection of Complex I Grim-19 (100 g mAb). . . . . . . . . . . . . . .. . . . .. .. . . . . . .. . $250
 MS105 Blue Native, Western Blotting and Immunocytochemistry Detection of Complex I 20kDa (100 g mAb). . . . . . . . . .. . . . . . .. . . . .. .. . . . . . .. . $250
 MS111 Blue Native, Western Blotting and Immunocytochemistry Detection of Complex I NDUFA9 (100 g mAb) . . . . . . . . . . .. . . .. . . . . . . . . . . . . $250
 MS204 Blue Native, Western Blotting and Immunocytochemistry Detection of Complex II-70 kD (flavoprotein) (100 g mAb). .. . . . . . . . . . . . . . . . .$250
 MS304 Blue Native, Western Blotting Detection of Complex III Core-2 (100 g mAb). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .$250
 MS407 Blue Native, Western Blotting and Immunocytochemical Detection of Complex IV COX IV (100 g mAb). . . . . . . . . . .. . . . . . . . . . . . . . . . . .$250
 MS408 Blue Native, Western Blotting and Immunocytochemical Detection of Complex IV COX IV (100 g mAb) . . . . . . . . . .. . . . . . . . . . . . . . . . . . $250
 MS507 Blue Native, Western Blotting and Immunocytochemistry Detection of Complex V -subunit (100 g mAb) . . . . . . . . . .. . . . . . . . . . . . . . . . $250

All MitoSciences products are sold “FOR RESEARCH PURPOSES ONLY”
MitoSciences LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801
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