Analytical Biochemistry 389 (2009) 143–149

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Resolving mitochondrial protein complexes using nongradient blue native

polyacrylamide gel electrophoresis
Liang-Jun Yan *, Michael J. Forster
Department of Pharmacology and Neuroscience and Institute for Aging and Alzheimer’s Disease Research, University of North Texas Health Science Center at Fort Worth,
Fort Worth, TX 76107, USA

a r t i c l e i n f o a b s t r a c t

Article history: Blue native polyacrylamide gel electrophoresis (BN–PAGE) is a powerful technique for separation and
Received 12 February 2009 proteomics analysis of high-molecular-weight protein complexes. It is often performed on gradient gels
Available online 5 April 2009 and is widely used for studying mitochondrial membrane complexes involved in electron transportation
and oxidative phosphorylation. In this article, we present an alternative BN–PAGE method that uses
Keywords: highly porous, nongradient polyacrylamide gels for separation of rat brain mitochondrial protein com-
Blue native polyacrylamide gel plexes. Results demonstrate that this method not only resolves mitochondrial complexes I to V, allowing
electrophoresis
subsequent analysis by in-gel activity staining and mass spectrometry peptide sequencing, but also iden-
Dihydrolipoamide dehydrogenase
Mitochondria
tifies Hsp60 polymers and dihydrolipoamide dehydrogenase (DLDH). Moreover, with this new method, it
Protein complexes is shown for the first time that complex I and DLDH can be detected simultaneously on a single gel strip
by in-gel activity staining. Overall, the method provides a simplified, nongradient gel electrophoretic
approach that should be useful in functional proteomics studies.
Ó 2009 Elsevier Inc. All rights reserved.

Blue native polyacrylamide gel electrophoresis (BN–PAGE)1 is a for diagnosing mitochondrial defects that are associated with human
powerful technique for isolation, separation, and detection of high- diseases [9–12].
molecular-weight protein complexes [1]. Two key features of BN– Conventionally, gradient BN–PAGE has been performed for the
PAGE facilitate the separation of protein complexes in their native separation of mitochondrial oxidative phosphorylation complexes
state without loss of enzymatic activity. The first one is the introduc- [13,14]. Although the gradient gel method is excellent for separa-
tion of Coomassie brilliant blue (CBB) G-250 into the protein sam- tion and detection of the five mitochondrial complexes that are in-
ples, a procedure that produces a surface negative charge shift that volved in oxidative phosphorylation, other mitochondrial protein
does not inactivate the proteins yet allows the proteins and their complexes are not detected under these conditions. For example,
complexes to resolve in the gel according to their native molecular both complex I and dihydrolipoamide dehydrogenase (DLDH) can
masses, their net surface charges, and their molecular shapes [2,3]. be stained using NADH and nitro blue tetrazolium (NBT) [15], yet
The second feature is the use of aminocaproic acid in sample prepa- the two have never been detected simultaneously on the same
ration and as a gel buffer component, which improves the solubiliza- gel strip. Gradient BN–PAGE used to isolate complex I usually fails
tion of membrane proteins [1]. Since its initial application for the to resolve a DLDH homodimer band; conversely, the nongradient
separation of mitochondrial membrane complexes during the early BN–PAGE method reported recently for detection of DLDH does
1990s [1], BN–PAGE has gained popularity and has been used suc- not isolate complex I [15]. In the current study, we provide an
cessfully for separation and analysis of mitochondrial and nonmitoc- alternative BN–PAGE approach that involves nongradient gel elec-
hondrial protein complexes [4], including nuclear protein complexes trophoresis on highly porous polyacrylamide gels. This technique
[5], water-soluble proteins [6,7], and whole cellular lysates [8]. yields, on the same gel strip, DLDH, Hsp60, and a separation of
Moreover, BN–PAGE has been applied successfully in clinical settings mitochondrial complexes I to V that are amenable to further anal-
ysis by in-gel activity measurements and mass spectrometry pep-
tide sequencing.
* Corresponding author. Fax: 817-735-0408.
E-mail address: [email protected] (L.-J. Yan).
1
Abbreviations used: BN–PAGE, blue native polyacrylamide gel electrophoresis; Materials and methods
CBB, Coomassie brilliant blue; DLDH, dihydrolipoamide dehydrogenase; NBT, nitro
blue tetrazolium; Bis, bis-acrylamide; DATD, N,N-diallytartardiamide; SDS, sodium Animals and chemicals
dodecyl sulfate; BSA, bovine serum albumin; CN–PAGE, clear native polyacrylamide
gel electrophoresis; DMSO, dimethyl sulfoxide; DAB, 3,30 -diaminobenzidine tetra-
chloride; MS/MS, tandem mass spectrometry; MALDI–TOF, matrix-assisted laser Tissues from adult Sprague–Dawley rats obtained from Harlan
desorption/ionization time-of-flight. (Indianapolis, IN, USA) were used to isolate mitochondria. These

0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2009.03.043
144 Nongradient blue native PAGE / L.-J. Yan, M.J. Forster / Anal. Biochem. 389 (2009) 143–149

experiments were conducted in adherence with the National Insti- SDS, and 10 mM glycerol [21] and were placed onto SDS–PAGE for
tutes of Health guidelines for the care and use of laboratory animals electrophoresis. Where indicated, clear native PAGE (CN–PAGE)
and were approved by the University of North Texas Health Science [22] was run under the same conditions except that the initial
Center animal care and use committee. The reagents and chemicals cathode buffer did not have Serva blue G-250. All gel images were
used for in-gel enzyme activity staining were purchased from Sigma documented using an Epson Perfection 1670 scanner.
(St. Louis, MO, USA) unless otherwise stated. Acrylamide, bis-acryl-
amide (Bis), N,N’-Diallytartardiamide (DATD), ammonium persul- In-gel enzyme activity staining
fate, and CBB G-250 were purchased from Bio-Rad Laboratories
(Hercules, CA, USA). Tricine and e-amino-N-caproic acid were pur- In-gel enzyme activity assays were performed for mitochondrial
chased from MP Biochemicals (Irvine, CA, USA). Bis–Tris was pur- complexes I to V and DLDH. All steps were conducted at room tem-
chased from Calbiochem (La Jolla, CA, USA). Serva blue G-250 was perature, with reactions being stopped at various time points by
obtained from Serva (Heidelberg, Germany). Protease inhibitor fixing the gel for 30 min in a solution containing 50% (v/v) metha-
cocktail tablets were purchased from Roche (Mannheim, Germany). nol and 10% (v/v) acetic acid. This was followed by long-term pres-
Prestained sodium dodecyl sulfate (SDS)–PAGE markers were ob- ervation of the gel in a solution containing 10% methanol and 8%
tained from Fermentas Life Sciences (Hanover, MD, USA). acetic acid at 4 oC.
For complex I and DLDH activity staining, the gel strip was incu-
Isolation of mitochondria and preparation of mitochondrial extracts bated in 50 ml of 50 mM potassium phosphate buffer (pH 7.0) con-
for BN–PAGE taining 0.2 mg/ml NBT and 0.1 mg/ml NADH [15]. For complex II
staining, the gel strip was incubated in 20 ml of 5 mM Tris–HCl
Mitochondria were isolated from whole rat brain using Percoll (pH 7.4) containing 0.5 M sodium succinate, 215 mM phenazine
gradient centrifugation [15]. Preparation of total mitochondrial ex- methosulfate (stock solution prepared in dimethyl sulfoxide
tracts for BN–PAGE analysis was performed as described previ- [DMSO]) and 20 mg of NBT [23]. For complexes III and IV staining,
ously [16] with modifications. Basically, mitochondrial pellet the gel strip was incubated in a 50-ml solution containing 50 mM
(either fresh or frozen) was resuspended at a protein concentration sodium phosphate (pH 7.2), 20 mg of 3,30 -diaminobenzidine tetra-
of approximately 1 mg/ml in a solubilization buffer containing chloride (DAB), and 50 mg of cytochrome c [23]. Under our gel
50 mM Bis–Tris (pH 7.0), 1% (v/v) n-dodecyl-b-d-maltoside, and electrophoretic conditions, we found that complex IV, but not com-
750 mM e-amino-N-caproic acid. The suspension was kept on ice plex III, could also be stained by incubating the gel strip in the
for 1 h with occasional vortexing and was then clarified by centri- above solution that did not contain cytochrome c, although it usu-
fugation at 20,000g for 30 min. Following the centrifugation, 0.9 ml ally took more than 6 h for the color to develop. Finally, for com-
of the resulting supernatant, containing both membrane and plex V staining, the gel strip was incubated in a 50-ml solution
water-soluble proteins, was mixed with 0.1 ml of concentrated containing 35 mM Tris, 270 mM glycine (pH 8.3), 14 mM MgCl2,
BN–PAGE loading buffer (10) containing 0.75 M e-amino-N-ca- 0.2% Pb(NO3)2, and 8 mM ATP [24]. This method of complex V
proic acid and 3% (w/v) Serva blue G-250 [15]. The samples were activity staining is based on the fact that the inorganic phosphate,
then stored at 20 °C until analysis. All protein concentrations originated from ATP hydrolysis catalyzed by complex V, reacts
were determined by bicinchoninic acid protein assay [17] using with lead nitrate to form lead phosphate that then accumulates
bovine serum albumin (BSA) as the standard. on the enzyme’s band [24].

Nongradient BN–PAGE SDS–PAGE and Western blots

An acrylamide/Bis solution that would yield highly porous gels Second-dimensional SDS–PAGE was performed according to
on polymerization was prepared as described previously [18,19] Laemmli [25] using a Bio-Rad Mini-PROTEAN III electrophoresis
with modifications. Essentially, a stock solution containing 50% cell. Both the stacking and resolving gels (4 and 10%, respectively)
(w/v) acrylamide and 0.5% (w/v) Bis (acrylamide/Bis = 100:1, w/ were made from a 30% acrylamide/Bis (29:1, w/w) solution. Usu-
w) was prepared in deionized distilled water and used for both ally, two gels were run simultaneously: one for protein staining
stacking and resolving gels. The final concentration of the stacking and the other for Western blot detection. After SDS–PAGE, gels
gel was 4%, and that of the resolving gel was 8% except where spec- were transferred to Hybond-C membranes with a Mini-Trans-Blot
ified. Nongradient BN–PAGE was performed at room temperature electrophoretic transfer cell (Bio-Rad) according to Towbin and
using a Bio-Rad Mini-PROTEAN III electrophoresis cell as described coworkers [26] with some modifications [27]. Western blots were
previously [15]. Gel buffer was composed of 500 mM aminocaproic performed according to the procedure described previously [21].
acid and 50 mM Bis–Tris (pH 7.0). Cathode buffer contained 50 mM
tricine, 15 mM Bis–Tris (pH 7.0), and 0.02% (w/v) Serva blue G-250, Protein identification by mass spectrometry peptide sequencing
and anode buffer contained 50 mM Bis–Tris (pH 7.0). Sample buffer
was 75 mM aminocaproic acid (final concentration) containing Protein identification was performed at ProtTech (Norristown,
0.3% (w/v) Serva blue G-250 (final concentration). Following sam- PA, USA) by using the NanoLC–tandem mass spectrometry (MS/
ple loading (20–30 lg proteins), the gel was run at 150 V until the MS) peptide sequencing technology. Briefly, a given blue native
front line had entered into one-third of the gel, and then the cath- gel band was destained, cleaned, and in-gel digested with sequenc-
ode buffer was replaced by the one that did not have Serva blue G- ing-grade trypsin. The resulting peptide mixture was analyzed by
250 (50 mM tricine and 15 mM Bis–Tris, pH 7.0). Gel running was an LC–MS/MS system in which a high-pressure liquid chromatog-
then continued at 200 V until complete. The stacking gel was then raphy device with a reverse-phase C18 column (75 lm i.d.) was
carefully removed using a blade prior to in-gel activity staining. coupled on-line with an ion trap mass spectrometer. The collected
Where needed, gels were stained by CBB G-250 [20], followed by mass spectrometric data were used to search the most recent non-
destaining in a solution containing 10% (v/v) methanol and 8% (v/ redundant protein database using ProtTech’s proprietary software
v) acetic acid. For BN–PAGE gel strips that were further processed suite. In contrast to matrix-assisted laser desorption/ionization
by second-dimensional SDS–PAGE, the gel strips, without fixing time-of-flight (MALDI–TOF)-based peptide mapping, the results
and staining, were equilibrated for 20 min in a solution containing from LC–MS/MS are based on independent peptide sequencing.
5% (v/v) 2-mercaptoethanol, 62.5 mM Tris–HCl (pH 6.8), 2.3% (w/v) In these studies, only those proteins that were confirmed by two
 Nongradient blue native PAGE / L.-J. Yan, M.J. Forster / Anal. Biochem. 389 (2009) 143–149 145

or more peptides sequenced (>99.9% certainty of identification) are staining developed at a much later time that was usually more
reported. Hence, all proteins identified in the gel bands represent than 15 min into the incubation. In addition, a DLDH band was
confirmed proteins rather than candidates. Moreover, the number usually not visible by CBB staining and could be visualized and
of peptides sequenced by LC–MS/MS from each protein can be used localized only on activity staining. Taken together, these results
as an indicator of their relative abundance in a mixture [28–30]. indicate that, in the mitochondria, complex I content is much
greater than DLDH content. We also tested the resolution of com-
Results plex I and DLDH using CN–PAGE [23], where Serva blue G-250 was
not included in the initial cathode running buffer, although the
Resolution of mitochondrial protein complexes loading buffer did contain Serva blue G-250. The result in Fig. 2B
shows that DLDH ran much slower on a clear native gel than on
To establish the conditions of nongradient BN–PAGE for resolv- a blue native gel, and the DLDH activity band was also more diffuse
ing mitochondrial protein complexes, we tested a series of gel con- on the clear native gel. These results suggest that BN–PAGE is bet-
centrations ranging from 7.5% to 12%. Fig. 1 shows protein band ter than CN–PAGE for DLDH separation and detection.
patterns at each acrylamide concentration. A comparison of the
patterns with those resolved by gradient BN–PAGE [1,23,24] indi- In-gel activity staining of complexes II to V
cates that mitochondrial complexes I and V, the two largest com-
plexes involved in oxidative phosphorylation, were always We next tested the feasibility of the nongradient electropho-
recognizable after gel electrophoresis under our experimental con- retic method for in-gel activity detection of mitochondrial com-
ditions (Fig. 1, as indicated on the 7.5% gel). For a clear visualiza- plexes II through V. Fig. 3A shows complex II activity staining
tion of other well-resolved protein bands, it was necessary to using sodium succinate as the substrate and NBT as the electron
further stain the gels with CBB G-250 followed by destaining. On acceptor in the presence of phenazine methosulfate [23]. Complex
destaining and storage, gels usually showed swelling that exhib- II is the smallest (130 kDa) among the five mitochondrial oxida-
ited approximately a 20% increase in gel area. tive phosphorylation complexes [31]. Hence, on an 8% gel, complex
As shown in Fig. 1, between 7.5% and 9.0% gels, complexes I and II was at the bottom of the gel and appeared to be diffuse (Fig. 3A,
V were well separated from each other, as were smaller protein left lane). On a 12% gel, however, a well-defined activity band of
complexes. Gel concentrations P 10%, however, yielded a progres- complex II could be visualized (Fig. 3A, right lane). Under these
sively poorer separation of complex I and/or complex V. For exam- experimental conditions, the activity staining usually took approx-
ple, on the 10% gel, complex I did not show a good separation and imately 30 min. It should be noted that for the 8% gel, it was nec-
complex V did, whereas on the 12% gel, neither complex I nor com- essary to remove the front-line Coomassie blue by destaining so
plex V exhibited a clear-cut separation (Fig. 1). In general, on a 7.5% that the color of NBT formazan could be highlighted.
gel, native proteins with molecular weight as low as 140 kDa could Under our experimental conditions, both complexes III and IV
be separated, whereas on a 12% gel, native proteins with molecular (along with an upper band indicated as supercomplex III) were
weight as low as 60 kDa could be resolved. It should be noted that stained by DAB/cytochrome c (Fig. 3B, lane 2). The color usually
on a 7.5% gel, complex II ( 130 kDa) could run out of the gel if the developed within 40 min of incubation. When the gel strip was
running was not stopped at the time when the front line (CBB G- incubated with DAB for a prolonged period (6–12 h) in the absence
250) reached the bottom of the gel. Taken together, these results of cytochrome c, only complex IV could be stained (Fig. 3B, lane 1)
indicated that 8 to 9% gels would be appropriate for resolving mito- and the activity band appeared to be less diffuse when compared
chondrial membrane complexes I to V. with that stained by DAB/cytochrome c (Fig. 3B, lane 2). The ratio-
nale for naming the upper band as supercomplex III was based on
Simultaneous staining of complex I and DLDH activity the following three observations: (i) the band ran slower than the
usual complex III band (Fig. 3B, lane 2); (ii) the band was stained by
Fig. 2A shows in-gel activity staining for both complex I and DAB/cytochrome c that could also stain the authentic complex III
DLDH on an 8% blue native gel. Under our experimental conditions, band (immediately below complex V in Fig. 3B, lane 2); and (iii)
complex I activity staining usually developed within 2 min of incu- when analyzed by NanoLC–MS/MS peptide sequencing, the band
bation in the presence of NADH and NBT, whereas DLDH activity was found to contain comparatively abundant complex III sub-

Gel concentration (%)

KDa
7.5 8.0 8.5 9.0 10 12
KDa 669
440
Complex I
669
232
Complex V

440 140
232

140 67

Fig. 1. Nongradient BN–PAGE resolution of total mitochondrial extracts. Shown are gels ranging from 7.5% to 12% stained with Coomassie blue following gel electrophoresis,
with 25 lg of protein being loaded in each lane. Mitochondrial extracts were prepared as described in the text in a BN–PAGE sample buffer containing 50 mM Bis–Tris (pH
7.0), 1% (v/v) n-dodecyl-b-d-maltoside, and 750 mM e-amino-N-caproic acid. Complexes I and V are indicated by arrows on the 7.5% gel. Native gel protein markers used in
this figure, as well as in other figures where indicated, are as follows: thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 232 kDa; lactate dehydrogenase, 140 kDa; BSA,
67 kDa.
146 Nongradient blue native PAGE / L.-J. Yan, M.J. Forster / Anal. Biochem. 389 (2009) 143–149

nongradient BN–PAGE was similar to that resolved by gradient

GE
GE
BN–PAGE [23].

- PA
- PA

CN
BN
Analysis of DLDH-associated protein components

Complex I Complex I In connection with our studies of DLDH oxidative modifications

[15,32], we were interested in investigating what proteins might
associate with DLDH. For this purpose, the band exhibiting DLDH
DLDH
activity, such as the one shown in Fig. 2A, was excised and ana-
lyzed by NanoLC–MS/MS peptide sequencing. Results in Table 1
show that a total of 24 proteins, including DLDH, were found to
be contained in the DLDH band. Based on the establishment that
DLDH the number of peptides sequenced from each protein usually re-
flects abundance of the protein in a sample [28–30], aconitase (Ta-
ble 1, no. 2), an enzyme in the Krebs cycle, was the major protein
that was associated with DLDH. Furthermore, other Krebs cycle en-
zymes such as malate dehydrogenase (no. 3), the complex II en-
zyme succinate dehydrogenase (no. 8), and citrate synthase (no.
9) were also found to be comparatively abundant in the DLDH
A B band. These findings should not be surprising given the fact that
DLDH is the E3 component of the a-ketoglutarate dehydrogenase
Fig. 2. Simultaneous in-gel activity staining of complex I and DLDH on a single gel complex that operates in the Krebs cycle. In addition, the data
strip. Following gel electrophoresis (8% resolving gel for both lanes A and B), each are also suggestive that DLDH is associated with mitochondrial
gel strip was incubated in 50 ml of 50 mM potassium phosphate buffer (pH 7.0)
containing 0.2 mg/ml NBT and 0.1 mg/ml NADH [15]. Shown are the resolutions of
membrane proteins, including ADP/TP translocase 1 (no. 6) and
complex I and DLDH by nongradient BN–PAGE (A) and CN–PAGE (B). complex V (no. 16). Indeed, the pyruvate dehydrogenase complex,
of which DLDH is also the E3 component, is known to be associated
with mitochondrial membranes [33–35]. Interestingly, none of the
units, including ubiquinol cytochrome c reductase core proteins 1 complex I subunits could be detected in the DLDH-containing
and 2 (see supplemental Table S1, nos. 3 and 6, in supplementary band, suggesting that complex I is not associated with DLDH. Like-
material). It should be noted that the presence of a trace amount wise, when the complex I band was analyzed by mass spectrome-
of three complex IV subunits in this band (see supplemental Table try peptide sequencing, DLDH was not among the proteins that
S1, nos. 7, 13, and 14) would seem to be unrelated to its complex III were identified (see supplemental Table S3 in supplementary
activity staining because the true complex III band did not contain material). Moreover, when a whole blue native gel strip was ana-
any detectable complex IV subunits (see supplemental Table S2) lyzed by second-dimensional SDS–PAGE in conjunction with Wes-
yet was stained by DAB/cytochrome c. tern blots probed with anti-DLDH antibodies, no DLDH signal was
The staining of complex V by lead phosphate took approxi- detectable at the locations where complex I subunits were ex-
mately 60 min and resulted in two bands showing complex V pected to resolve (Fig. 4). In any case, these results indicate that
activity (Fig. 3C). As reported previously [23,24], and as indi- DLDH is not associated with complex I, and this may explain
cated in Fig. 3C, these two bands represented the ATP hydroly- why DLDH is not susceptible to oxidative attacks by complex I-
sis activity of holocomplex V and ATP hydrolysis activity of the generated reactive oxygen species (our laboratory, unpublished
F1 subcomplex. Therefore, the pattern of complex V resolved by work).
to c
+ cy
DAB
DAB
%
8%

12

Complex I
Supercomplex III
Complex V Complex Va (Holo-F1F0 complex)
Complex III
Complex II
Complex Vb (F1 subcomplex)
Complex IV
Complex II

1 2
A B C
Fig. 3. In-gel activity staining of complexes II, III, IV, and V. Except where indicated, all gels were 8%. (A) Complex II activity staining on 8% (left lane) and 12% (right lane) blue
native gel strips. Note that Coomassie blue at the bottom of the 8% gel needed to be removed by destaining of the gel following activity staining so that a better complex II
activity staining signal (NBT formazan) could be observed. (B) Activity staining of complexes III and IV. Under our gel electrophoresis conditions, DAB stained only complex IV
(lane 1), whereas DAB and cytochrome c stained both complexes III and IV along with an upper band indicated as supercomplex III (lane 2). (C) Activity staining of complex V.
Note that for a better visual effect, the color of the gel image was inverted to highlight complex V activity staining.
 Nongradient blue native PAGE / L.-J. Yan, M.J. Forster / Anal. Biochem. 389 (2009) 143–149 147

Table 1
Proteins identified in DLDH-associated gel band resolved by nongradient BN–PAGE.

Protein name MW (Da) Access number (NCBI) Number of peptides sequenced

1. Dihydrolipoamide dehydrogenase 54574.33 40786469 15
2. Aconitase 86121.31 40538860 13
3. Malate dehydrogenase 36116.98 42476181 7
4. Glutamate oxaloacetate transaminase 2 47683.27 6980972 6
5. 4-Aminobutyrate aminotransferase 57159.82 13591900 6
6. ADP/ATP translocase 1 33196.32 32189355 5
7. Hexokinase 1 103539.66 6981022 5
8. Succinate dehydrogenase complex, subunit A, flavoprotein 72596.11 18426858 5
9. Citrate synthase 52175.60 18543177 4
10. Aldolase A 39783.44 6978487 4
11. Tubulin, alpha 1B 50803.87 34740335 4
12. Pyruvate carboxylase 130349.04 929988 4
13. Neuron-specific class III beta tubulin 53599.25 20799322 4
14. Aldehyde dehydrogenase, family 6, subfamily A1 58223.79 145651820 3
15. Predicted: similar to histone H2A type 1 18550.45 109505989 3
16. ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 59830.73 40538742 3
17. Acetyl-coenzyme A acetyltransferase 1 45022.61 8392836 3
18. Pyruvate dehydrogenase 39299.18 56090293 3
19. Nicotinamide nucleotide transdehydrogenase 114537.59 61557127 3
20. Amphiphysin 1 74946.18 11560002 2
21. Aldolase C 39658.33 6978489 2
22. Dynamin 1 96209.41 18093102 2
23. Predicted: similar to succinate dehydrogenase lp subuni 32607.22 109475694 2
24. Enoyl coenzyme A hydratase, short chain 1 31895.33 17530977 2

Note. Protein identification was carried out using the NanoLC–MS/MS peptide sequencing technique as described in the text. NCBI, National Center for Biotechnology
Information.

Overview of mitochondrial protein complexes resolved by nongradient

dria
BN–PAGE Pseudo-complex A

chon
er
Mark

Based on the results presented in Figs. 2 and 3 and the related Mito
NanoLC–MS/MS peptide sequencing results as described immedi- Pseudo-complex B
kDa
ately below, the identity of each band on an 8% nongradient BN–
Complex I
PAGE is given in Fig. 5. The very top two bands were labeled as 669 Supercomplex III*
pseudo-complexes A and B because each band was found to con- Complex Va (holo-F1F0 complex)
tain many different proteins that appeared to migrate together un- Complex III
der these particular electrophoretic conditions (see supplemental b Hsp 60
440
Tables S4 and S5 in supplementary material). Band p, which also Complex Vb (F1 subcomplex)
appeared to contain miscellaneous comigrating proteins (see sup- DLDH*
232 p Pseudo-complex C
plemental Table S6), was labeled as pseudo-complex C. On the
other hand, mass spectrometry peptide sequencing results indicate Complex IV
that the predominant proteins contained in band b comprise
Hsp60 (see supplemental Table S7), as identified previously using Complex II
140
gradient BN–PAGE [36] or gradient CN–PAGE [22]. Taken together,
Fig. 5. Representative nongradient blue native gel image showing the resolution of
rat brain mitochondrial protein complexes. In addition to the five complexes
involved in oxidative phosphorylation, the band for Hsp60, the location of DLDH,
I V III DLDH and the bands containing miscellaneous proteins that comigrated are also indicated
(pseudo-complexes A, B, and C). Shown is the result of an 8% gel with 25 lg of
Mito * mitochondrial proteins that were loaded. *Labeling based on activity staining.
BN-PAGE
kDa
250 results of this study demonstrate that our nongradient BN–PAGE
130
95 approach is capable of resolving mitochondrial complexes I to V,
72 Hsp60, and DLDH.
55
DLDH
(positive control) 36 Discussion
Anti-DLDH blot
28 DLDH
In the current report, we have described a nongradient BN–
17
PAGE method for separation and analysis of mitochondrial protein
complexes. The establishment of the method was achieved mainly
Fig. 4. Two-dimensional Western blot probed with anti-DLDH antibodies. A BN– by increasing the ratio of acrylamide to Bis (100:1, w/w) that con-
PAGE gel strip (upper panel) was equilibrated for 20 min in a solution containing 5% sequently increased the pore size of the gel [18,19]. The results
(v/v) 2-mercaptoethanol, 62.5 mM Tris–HCl (pH 6.8), 2.3% (w/v) SDS, and 10 mM presented demonstrate that this nongradient BN–PAGE resolves
glycerol [21]. The gel strip was then placed on top of a 10% polyacrylamide gel for
SDS–PAGE, which was followed by standard Western blot (lower panel) procedures
not only all five known mitochondrial protein complexes involved
as described in the text. *Loading of mitochondrial extracts as a positive control for in oxidative phosphorylation but also other protein complexes
DLDH protein. such as DLDH and Hsp60 as well as three gel bands that contain
148 Nongradient blue native PAGE / L.-J. Yan, M.J. Forster / Anal. Biochem. 389 (2009) 143–149

miscellaneous proteins migrating together under these gel electro- complexes can also be detected histochemically. In agreement
phoretic conditions. with previous reports [23], we found that complex IV could be
Although Bis was used as the crosslinker in the current study for readily stained by DAB/cytochrome c. Interestingly, the same con-
the method development, the effect of another crosslinker, DATD, ditions (DAB/cytochrome c) also led to a discernible and relatively
on resolving mitochondrial protein complexes was also evaluated rapid staining of complex III. Mass spectrometry peptide sequenc-
during the studies. For a given ratio of acrylamide to each cross- ing of the complex III band failed to indicate the presence of any
linker, DATD is known to generate a gel with a larger pore size than complex IV subunits (see supplemental Table S2); thus, the reason
does Bis [37,38]. Indeed, when DATD was used as the crosslinker, why complex III enzymatic activity could be detected by DAB/cyto-
the gel concentrations could be moderately increased to achieve chrome c under our experimental conditions remains unclear.
a similar and comparable protein resolution. For example, protein These results may indicate that, under certain blue native gel elec-
band patterns resolved by a 10% gel made from an acrylamide/ trophoretic conditions, it is possible to detect complex III activity
DATD (100:1, w/w, 50%) solution were similar to those resolved using the DAB/cytochrome c staining system.
by an 8.0% gel made from the acrylamide/Bis (100:1, w/w) solution DAB itself, however, could not stain complex III activity under
(data not shown). In addition, in agreement with previous findings our experimental conditions. This is in disagreement with previous
[39,40], DATD-crosslinked gels were found to be elastic and sticky studies [23], where complex III separated by CN–PAGE was detect-
to glass and also to show swelling on storage. Under our experi- able using DAB. The reason for this disagreement is likely due to
mental conditions, the swelling of a 10% DATD gel could eventually the use of Serva blue G-250 in our system given that this could
exhibit approximately a 130% increase in gel area in destaining interfere with DAB staining of complex III activity [23]. On the
solution. Nevertheless, no band distortions occurred (data not other hand, in agreement with previous studies [24], complex IV
shown). One caveat associated with the use of DATD for gel elec- activity staining by either DAB alone (Fig. 3B, lane 1) or DAB/cyto-
trophoresis is that the 4% stacking gel must be made from the chrome c (Fig. 3B, lane 2) was found to be catalase independent
50% acrylamide/Bis (100:1, w/w) solution such that firmly formed (data not shown), as was complex III activity staining by DAB/cyto-
loading wells can be established. This is because a 4% acrylamide/ chrome c (Fig. 3B, lane 2). Hence, our results also ruled out the
DATD (100:1, w/w) solution did not polymerize under our experi- involvement of a basal level production of H2O2 that would other-
mental conditions, probably due to the combined effect of the low wise induce DAB polymerization and precipitation [42]. For in-gel
DATD concentration in the 4% solution and the presence of ambi- complex V activity detection, our results are similar to those re-
ent oxygen that could inhibit the gel polymerization. With due ported previously given that we also found that complex V was re-
consideration of the above caveats, DATD-crosslinked, nongradient solved into two bands, both of which possessed ATP hydrolysis
blue native gels (10% resolving) also work well for the analysis of activity [23,24] (Fig. 3C).
mitochondrial protein complexes. Finally, as expected, our method of nongradient BN–PAGE is
Recently, we reported a BN–PAGE method for DLDH isolation compatible with mass spectrometry analyses of proteins (Table 1
and detection [15]. However, neither the gel concentration (9%) and supplemental Tables S1–S7) that are of interest. In addition,
nor the pore size of the gel (acrylamide/Bis = 29:1, w/w) used in the gel strips generated by this method can also be further ana-
that study is conducive to complex I separation. We have found lyzed by second-dimensional SDS–PAGE for Western blot probing
that complex I can be separated on a 6% blue native gel (acrylam- of target proteins (Fig. 4). Moreover, with this new method, the iso-
ide/Bis = 29:1, w/w), but DLDH fails to be detected on such a gel lation of much smaller native proteins or protein complexes
(data not shown), which is also too fragile for handling. Hence, (<100 kDa), including those of cytosolic and nuclear proteins,
nongradient BN–PAGE made from a 29:1 (w/w) acrylamide/Bis should be technically possible by increasing the acrylamide con-
solution [15] cannot accommodate complex I and DLDH simulta- centration of a nongradient blue native gel (e.g., the 12% gel in
neously. In contrast, through increasing the pore size of the gels Fig. 1).
as described in the current study (acrylamide/Bis = 100:1, w/w), In summary, a nongradient BN–PAGE method for separation
we showed for the first time that complex I and DLDH can indeed and histochemical staining of mitochondrial complexes has been
be detected simultaneously on the same gel strip. presented. This method can be used as an alternative simplified
The results of the current study, together with previous obser- technique for isolating mitochondrial protein complexes and
vations [15,32], indicate that the successful separation of DLDH other cellular protein complexes for functional proteomics
by BN–PAGE can be attributed to at least two factors. First, an ac- studies.
tive form of DLDH is a stable homodimer [32,41]. The DLDH
homodimer is, in fact, so stable that the presence of 2 M urea in Acknowledgments
the gel failed to disrupt the DLDH homodimeric state (data not
shown). Second, the presence of Serva blue G-250 in the loading This work was supported by award PO1AG022550 from the Na-
sample and in the initial cathode running buffer greatly facilitates tional Institute on Aging. The content is solely the responsibility of
the separation of DLDH. Indeed, when mitochondrial protein ex- the authors and does not necessarily represent the official views of
tracts were analyzed by CN–PAGE, where Serva blue G-250 was the National Institute on Aging or the National Institutes of Health.
omitted, a poor separation of DLDH from complex I occurred The authors thank Drake Zhang at ProtTech for his assistance in
(Fig. 2B), demonstrating that Serva blue G-250 acts as a driving mass spectrometry peptide sequencing.
force for DLDH separation during blue native gel electrophoresis.
It should be noted that with the current nongradient gel approach,
Appendix A. Supplementary data
DLDH can be located and identified only by in-gel activity staining,
whereas with our previously reported method [15], DLDH can be
Supplementary data associated with this article can be found, in
visualized by either Coomassie blue staining or in-gel activity
the online version, at doi:10.1016/j.ab.2009.03.043.
staining. The reason for this discrepancy remains unclear, but gel
porosity and the respective detergents (n-dodecyl-b-D-maltoside
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