[Methods in Cell Biology] Mitochondria, 2nd Edition Volume 80 || Electrophoretic Methods to Isolate...
Transcript of [Methods in Cell Biology] Mitochondria, 2nd Edition Volume 80 || Electrophoretic Methods to Isolate...
CHAPTER 33
METHODS IN CELL BIOLCopyright 2007, Elsevier Inc.
Electrophoretic Methods to Isolate ProteinComplexes from Mitochondria
Ilka Wittig and Hermann SchaggerMolekulare Bioenergetik, ZBCUniversitatsklinikum Frankfurt60590 Frankfurt am Main, Germany
I. In
OGY,All rig
troduction
VOL. 80 0091hts reserved. 723 DOI: 10.1016/S0091
-679X-679X
II. M
aterials and Methods A. C hemicals B. F irst Dimension: Blue Native Electrophoresis C. F irst Dimension: Clear Native Electrophoresis D. S econd Dimension: Modified Blue Native Electrophoresis E. S econd or Third Dimension: SDS-PAGEIII. A
pplications A. P rotein Complexes from Mitochondria and Chloroplasts B. Is olation of Protein Complexes from Tissue Homogenates and Cell Lines C. P urification of Partially Purified Protein Complexes Using BN-PAGEIV. O
utlook R eferencesI. Introduction
Blue native electrophoresis (BNE), which was initially named blue native
polyacrylamide gel electrophoresis (BN-PAGE), was first described in 1991
(Schagger and von Jagow, 1991). The original protocols for BNE and related
techniques have been improved and expanded considerably, as summarized here.
As a basic modification, imidazole buVer has replaced Bis-Tris buVer, becauseit does not interfere with protein determinations. The basic principles underlying
the native electrophoretic techniques used, however, remained unchanged. Mild
nonionic detergents are used for solubilization of biological membranes for
/07 $35.00(06)80033-6
724 Ilka Wittig and Hermann Schagger
BNE. After solubilization, an anionic dye (Coomassie blue G-250) is added
which binds to the surface of all membrane proteins and many water-soluble
proteins.
This binding of a large number of dye molecules to solubilized proteins pro-
duces a number of eVects that are advantageous for BNE. First, the isoelectric
points (pI ) of the proteins are shifted to the acidic range. Therefore, all proteins
migrate to the anode and can be separated on the basis of their migration distance
in gradient polyacrylamide gels. Second, the negative charge of the dye on the
surface of proteins reduces their aggregation and converts membrane proteins into
water-soluble proteins. Therefore, once Coomassie dye is bound, any detergent can
be omitted from the gels. This minimizes the risk of denaturation of detergent-
sensitive membrane protein complexes. Third, proteins that bind Coomassie dye
are visible during electrophoresis and migrate as blue bands. This facilitates exci-
sion of bands and recovery of proteins and complexes in a native state by native
electroelution.
Novel applications of clear native electrophoresis (CNE; Wittig and Schagger,
2005) are also described. CNE diVers from BNE in that Coomassie dye is not
used in CNE. Therefore, proteins migrate in an electrical field according to two
parameters: the intrinsic pI of the protein and the actual running pH in the gel.
In CNE, which uses the imidazole buVer system, a pH of 7.5 develops in the run-
ning gel during electrophoresis. As a result, this type of CNE can only separate
proteins with pI<7.5. An alternative Tris buVer system (running at pH 8.7) can be
used to separate proteins with higher pI. CNE has lower resolution compared to
BNE but is the method of choice in cases where Coomassie dye interferes with
techniques required to further analyze native complexes, for example, in-gel deter-
mination of catalytic activities, fluorescence resonance energy transfer (FRET)
analyses, and in-gel detection of fluorescent markers in general (Gavin et al.,
2003, 2005). Careful choice of detergent and appropriate detergent/protein ratio
has helped to preserve some native protein–protein interactions during BNE,
including the dimeric state of mitochondrial ATP synthase (Arnold et al., 1998;
Schagger and PfeiVer, 2000). Using identical solubilization conditions but CNE
instead of BNE, even higher oligomeric states of ATP synthase, such as tetramers,
hexamers, and octamers, have been identified and separated (Wittig and Schagger,
2005).
Second dimension BNE (modified by adding detergent to the blue cathode
buVer) can follow first dimension CNE or first dimension BNE whenever gentle
dissociation of supramolecular assemblies into their constituent complexes is
desired (Schagger and PfeiVer, 2000). SDS-PAGE or doubled SDS-PAGE
(dSDS-PAGE; i.e., two orthogonal SDS-gels with strongly diVering gel types for
first and second dimensions) usually follow as final steps to separate the subunits
of complexes and to identify very hydrophobic membrane proteins by mass
spectrometry (Rais et al., 2004).
Table IBuVers for BNEa
Soluti
Cathode buVer Bb (d
Cathode buVer B/10
Gel buVer 3� (triple
Anode buVer
5% Coomassie blue
AB mix (49.5% T, 3%
acrylamide-bisacry
aImidazole is introbCathode buVer B
aggregates at low temcCathode buVer Bd6-Aminohexanoi
protease inhibitor. B
conditions.eT, total concentr
33. Electrophoretic Methods to Isolate Protein Complexes from Mitochondria 725
II. Materials and Methods
A. Chemicals
Dodecyl-b-D-maltoside, Triton X-100, digitonin (Cat. No. 37006, >50% purity,
used without recrystallization), and 6-aminohexanoic acid are from Fluka (Buchs,
CH, Switzerland). Acrylamide, bis-acrylamide (the commercial, twice-crystallized
products), and Serva Blue G (Coomassie blue G-250) are from Serva (Heidelberg,
Germany). All other chemicals are from Sigma (Buchs, CH, Switzerland).
B. First Dimension: Blue Native Electrophoresis
1. Detergents, Stock Solutions, and BuVers
In principle, any nonionic detergent or mild anionic detergent, such as cholic
acid derivatives, can be used for the solubilization of biological membranes for
BNE, as long as the detergent can solubilize the desired protein and keep it in the
native state. We prefer to use dodecyl-b-D-maltoside, Triton X-100, and digitonin,
all of which are stored as 1–20% stock solutions in water. Stock solutions of
Tricine (1M), 6-aminohexanoic acid (2M), imidazole (1M), and imidazole/HCl
(1M, pH 7.0) are stored at 7�C. BuVers for BNE are summarized in Table I.
on Composition
eep blue) 50-mM Tricine, 7.5-mM imidazole (the resulting pH is around 7.0),
plus 0.02% Coomassie blue G-250c (slightly blue) As above but lower dye concentration (0.002%)
concentrated) 75-mM Imidazole/HCl (pH 7.0), 1.5-M 6-aminohexanoic acidd
25-mM Imidazole/HCl (pH 7.0)
Suspended in 500-mM 6-aminohexanoic acid
C
lamide mixture)e48-g Acrylamide and 1.5-g bisacrylamide per 100 ml
duced instead of Bis-Tris because Bis-Tris interferes with many commonly used protein determinations.
is stirred for several hours before use and stored at room temperature, since Coomassie dye can form
perature that can prevent proteins from entering the gel.
/10 and all other solutions can be stored at 7�C.
c acid is not essential for BNE but it improves protein solubility and is an eYcient and inexpensive serine
N-gels can be stored at 4�C for several days. We did not observe protease degradation under these
ation of acrylamide and bisacrylamide monomers; % C, percentage of cross-linker to total monomer.
726 Ilka Wittig and Hermann Schagger
2. Gel Types
Gels that resolve specific molecular mass ranges are listed in Table II. Each of
these gels are 0.16�14�14 cm, and are used either as ‘‘analytical gels’’ (with 0.5- or
1.0-cm sample wells) or as ‘‘preparative gels’’ (with one 14-cm sample well). Gradi-
ent gel preparation is exemplified in Table III.
3. Sample Preparation
a. How Much Detergent Is Required for Complete Solubilization of Membrane Proteinsfrom Biological Membranes?
As a general rule of thumb, bacterial membranes require Triton X-100 or dode-
cylmaltoside at a detergent/protein ratio of 1 g/g for solubilization, but 2–3 g/g
is necessary for solubilization of mitochondrial membranes. Using digitonin, the
Table IIGel Types for BNE
Mass range (kDa)a Sample gel (% T) Gradient gel (% T)
10–10,000 3.0 3!13
10–3000 3.5 4!13
10–1000 4.0 5!13
10–500 4.0 6!18
aResolution in the 10- to 100-kDa range is low in BNE with all gel types.
Table IIIGradient Gel Preparationa
Sample gel Gradient separation gel
4% T 5% T 13% T
AB mix 0.5 ml 1.9 ml 3.9 ml
Gel buVer 3� 2 ml 6 ml 5 ml
Glycerol – – 3 g
Water 3.5 ml 10 ml 3 ml
10% APSb 50 ml 100 ml 75 mlTEMED 5 ml 10 ml 7.5 mlTotal volume 6 ml 18 ml 15 ml
aVolumes for one gel (0.16�14�14 cm). Linear gradient separation gels are
cast at 4�C and maintained at room temperature for polymerization. The volume
of the 5% T solution is greater than that of the 13% T solution containing glycerol.
This assures that the two solutions initially are not mixed when the connecting
tube is opened. The sample gel is cast at room temperature. After removal of the
combs, gels are overlaid with gel buVer 1� and stored at 4�C.
b10% Aqueous ammonium persulfate solution, freshly prepared.
33. Electrophoretic Methods to Isolate Protein Complexes from Mitochondria 727
detergent/protein ratio is doubled (e.g., 4–6 g/g for mitochondrial membranes) to
achieve comparable solubilization, since the mass of digitonin is about twice the
mass of Triton X-100 and dodecylmaltoside.
For solubilization pilot studies, use a series of detergent/protein ratios, for
example, 0.5, 1.0, 1.5, and 2.0 g dodecylmaltoside per gram of protein (for bacterial
membranes). This titration will reveal the eYciency of solubilization, and whether
the enzyme of interest is detergent sensitive with respect to its physiological agg-
regation state, catalytic activity, or subunit composition. For example, the enzyme
might be dimeric (active) using low-detergent but monomeric (inactive) using
high-detergent conditions. Retention or loss of detergent-labile subunits can be
analyzed by second dimension SDS-PAGE.
b. What Is the Maximal and Minimal Protein Load for BN-PAGE?As a general rule of thumb, the maximum protein load depends more on the
DNA content of the solubilized sample than on the protein quantity. Using
isolated mitochondria (low DNA), the maximum protein load for application to
a 0.16 � 1 cm sample well is 200–400 mg of total protein. Using bacterial mem-
branes directly (without removing DNA), the protein load should be reduced to
around 50–100 mg. DNA probably blocks the pores of the sample gel, which
prevents proteins from entering the gel. Application of DNases does not help.
In our hands, deep blue artifact bands appeared in gels of DNase-treated samples.
The best way to remove DNA is by mild disruption of cells and diVerentialcentrifugation.
The minimal load for BNE depends on the sensitivity of the protein detection
method. When specific antibodies are available there is essentially no minimal
protein load for BNE. However, the detergent/protein ratio used for solubilization
of small and large amounts of protein must be the same, and the detergent con-
centration thereby must be clearly above the critical micelle concentration (cmc).
The latter condition can be kept using low final volumes for low-protein amounts.
c. General Scheme for Solubilizing Biological MembranesBiological membranes usually are suspended with small volumes of carbohy-
drate- or glycerol-containing buVers (e.g., 250-mM sucrose, 400-mM sorbitol, or
10% glycerol). Aliquots of these samples can be shock-frozen in liquid nitrogen and
stored at –80 �C without dissociating multiprotein complexes. The salt concentra-
tion in these suspensions should be low (0- to 50-mM NaCl). Potassium and diva-
lent cations should be avoided because they can cause aggregation of Coomassie
dye and precipitation of Coomassie-associated proteins. The preferred buVer is
50-mM imidazole/HCl, pH 7.0. Other buVers, for example, Na-phosphate or
Na-MOPS, can be used at 5- to 20-mM concentrations. Concentrated protein sus-
pensions (>10 mg/ml) can be used directly for solubilization. Organelle or vesicle
suspensions with low-protein concentrations should be diluted and then concen-
trated by centrifugation. Finally, all solubilization steps are carried out on ice.
728 Ilka Wittig and Hermann Schagger
Procedure
1. Solubilize membrane pellets (50- to 400-mg sedimented protein) by adding
10–40 ml of solubilization buVer (50-mM NaCl, 50-mM imidazole/HCl, pH 7.0,
usually containing 5-mM 6-aminohexanoic acid and 1-mM EDTA) and deter-
gent (from 1% to 20% stock solutions) at predetermined detergent/protein ratios
(see Section II.B.3.a). Higher salt concentrations should be avoided, since high salt
can lead to stacking of proteins in the sample wells and highly concentrated
membrane proteins tend to aggregate. Solubilization is complete within several
minutes.
2. Centrifuge the sample for 10–30 min at 100,000 � g at 4 �C, depending on
the sample volume. In cases where large proteins complexes (e.g., >5-MDa com-
plexes) are analyzed, centrifuge solubilized samples at 20,000� g for 20min at 4 �C.The supernatant obtained from this centrifugation is applied to the BN-gel.
3. Prior to sample application, rinse the BN-gel wells to remove gel storage
buVer.
4. Add 5% glycerol to the supernatants to facilitate sample loading.
5. Shortly before loading the sample, add Coomassie dye from a 5% suspension
in 500-mM6-aminohexanoic acid. The amount of added dye depends on the amount
of detergent used. The optimum dye/detergent ratio is in the range from 1:4 to
1:10 g/g.
Note: Careful determination of the correct amount of dye to add is particularly
important with samples that require high-detergent concentrations, for example,
2–4%, for solubilization. Excess lipid/detergent micelles incorporate the anionic
dye and therefore migrate very fast. This removal of lipid/detergent/dye micelles
improves protein resolution. A mixture of nonionic detergent and anionic dye
can mimic some properties of an anionic detergent. For most multiprotein com-
plexes, this ‘‘anionic detergent’’ does not dissociate labile protein subunits,
because the extracted membrane protein complexes are suYciently shielded
by boundary lipid.
However, there are situations where addition of Coomassie dye to the sample is
not advisable, and clear samples or samples containing the red dye Ponceau S, as
in CNE, are preferred. For example, membrane protein complexes that are
purified by chromatographic protocols often contain reduced amounts of bound-
ary lipid. Detergent-labile subunits can dissociate from these partially delipidated
samples on addition of Coomassie dye.
4. Running Conditions
1. BNE usually is performed at 4–7 �C, as broadening of bands was observed at
RT, and cathode buVer B (Table I) is commonly used.
2. 100 V is applied until the sample has entered the gel. Thereafter, the current
is limited to 15 mA and voltage is limited to 500 V.
33. Electrophoretic Methods to Isolate Protein Complexes from Mitochondria 729
3. After ab out one -third of the total running distance, cathode buV er B isremove d, and the run is continued using c athode bu Ver B/10. (This buV er ex-change impro ves de tection of faint protein ban ds, helps with native blott ing, as
less Coom assi e dye co mpetes with protei n binding to PVDF membr anes, an d
improves the performan ce of SDS electro phoresi s in the seco nd dimens ion.)
4. Run times are typic ally 2–5 h.
5 . Electroelution of Native Proteins
Two points are essent ial for the elect roelution of native protei ns. First, a ban d
of the de sired pro tein (or at least a ba nd of a marker protei n in the vici nity)
must be detect able at the end of the run to faci litate excis ion of gel bands an d
electroelut ion of nativ e protei ns. Secon d, since protein s stop migr ating co mpletely
when they approac h a mass -specifi c pore size lim it, it is essent ial that BN E is
terminat ed early , for exampl e, after half of the normal ru nning dist ance. At this
stage, the proteins are still mobile in the BN-gel, and can be efficiently extracted
from the gel using H-sh aped elutor vessel s bui lt accordi ng to Hu nkapiller et al .
(1983).
Procedure
1. Seal both lower ends of the elutor vessel with dialysis membranes with a
low-cutoff value, for example 2 kDa. (We find that low-cutoff dialysis membranes
are more mechanically stable compared to other dialysis membranes (Schagger,
2003a,b).
2. Excise blue protein bands from the gel, mash the gel by several passages
through 1-ml syringes, and transfer the mashed gel to the cathodic arm of the H-
shaped elutor vessel.
3. Fill both arms of the chamber and the horizontal connecting tube with
electrode buVer (25-mM Tricine; 3.75-mM imidazole, pH 7.0; and 5-mM 6-amino-
hexanoic acid (as a protease inhibitor).
4. Extract for several hours using 500 V with current that is limited to 2 mA per
elutor vessel (to prevent damage if a high-salt buVer was used erroneously). Partially
aggregated proteins are collected as a blue layer on the anodic dialysis membrane.
To avoid protein aggregation, the voltage can be reduced to 200 V.
6. Semidry Electroblotting of Native Proteins
Similar to native electroelution, native electroblotting also requires short runs of
BNE for the eYcient transfer of proteins (see earlier discussion). Use cathode buVerB/10 and not cathode buVer B as the BNE cathode buVer before electroblotting,
because Coomassie dye binds to PVDFmembranes, reducing their protein-binding
capacity. Do not use nitrocellulose membranes because they cannot be destained
using the conditions described below.
Table IVBuVers for CNE (Running pH Around 7.5)
Solution Composition
Cathode buVer 50-mM Tricine, 7.5-mM imidazole (resulting pH is around 7.0 at 4�C)
Gel buVer 3� (triple
concentrated)
75-mM Imidazole/HCl (pH 7.0; 4�C), 1.5-M 6-aminohexanoic acid
Anode buVer 25-mM Imidazole/HCl (pH 7.0; 4�C)
Glycerol/Ponceau S solution 50% Glycerol, 0.1% Ponceau S
AB mix (49.5% T, 3% C) 48-g Acrylamide and 1.5-g bisacrylamide per 100 ml
730 Ilka Wittig and Hermann Schagger
Procedure
1. Place a stack of 4 sheets of Whatman 17 CHR filter papers (total width is
3 millimeters) that has been wetted with electrode buffer on the lower electrode of
the semi-dry blotting apparatus (the cathode in this arrangement). The electrode
buffer is the cathode buffer for CNE, Table IV; 50-mM Tricine, 7.5 mM imidaz-
ole; the resulting pH is around 7.0.
2. Place the gel and then the PVDF membrane, for example, Immobilon PÒ
(wetted with methanol and soaked in electrode buffer) on the filter paper stack.
Then put another stack of wetted sheets of filter papers on top, mount the anode
of the electroblotting apparatus, and finally place a 5 kg load on top.
3. The transfer is at 4 �C (which is recommended) or at RT, using voltage that is
set to 20 V (actual voltage around 7 V) and current that is limited to 0.5 mA/cm2
(80 mA for a 12�14 cm2 gel area). The transfer is usually complete after 3 h.
4. To destain the background, incubate membrane in 25% methanol, 10%
acetic acid.
C. First Dimension: Clear Native Electrophoresis
A basic version of CNE, originally termed colorless native PAGE (CN-PAGE;
Schagger et al., 1994), was described shortly after the development of BNE (Schagger
and von Jagow, 1991). Advantages and limitations of CNE have been discussed
(Wittig and Schagger, 2005). The resolution of CNE is clearly lower compared to
BNE (Fig. 2A). However, CNE is the method of choice when Coomassie dye used in
BNE interferes with techniques required to further analyze native proteins.
The first step in CNE and BNE is identical, that is, biological membranes are
solubilized under the same ionic strength, pH, and detergent conditions. Dode-
cylmaltoside is the common detergent for the separation of individual complexes,
and digitonin is used for the isolation of larger physiological assemblies. Fol-
lowing clarification of solubilized sample by centrifugation, add 1 volume of a
Table VBuVers for CNE (Running pH Around 8.7)
Solution Composition
Cathode buVer 100-mM glycine, 15-mM Tris (resulting pH is around 8.7 at 4�C)
Gel buVer 3� (triple concentrated) 75-mM Tris–HCl (pH 8.5; 4�C), 1.5-M 6-aminohexanoic acid
Anode buVer 25-mM Tris–HCl (pH 8.4; 4�C)
Glycerol/Ponceau S solution 50% Glycerol, 0.1% Ponceau S
AB mix (49.5% T, 3% C) 48-g Acrylamide and 1.5-g bisacrylamide per 100 ml
33. Electrophoretic Methods to Isolate Protein Complexes from Mitochondria 731
solution consisting of 50% glycerol and 0.1% Ponceau S to 9 volumes of solubi-
lized sample. The red dye Ponceau S facilitates loading the sample (around 20 mlper 0.16 � 0.5 cm sample well) and marks the running front but does not bind to
proteins.
BuVers required for CNE are summarized in Tables IV and V. Using the
imidazole–Tricine system (Table IV), the running pH is around 7.5. This CNE system
is useful for proteins with pI< 7.5 thatwillmigrate toward the anode. The alternative
Tris–glycine system (Table V), where the running pH is around 8.7, may be used for
proteins with pI < 8.7. We have not tried to increase the running pH beyond 8.7
because multiprotein complexes may dissociate when exposed to high pH.
Gel types for BNE (Table II) can also be used for CNE with minor modifica-
tions. (1) When dodecylmaltoside is used for membrane solubilization, 0.03%
dodecylmaltoside should be added to the gradient gel mixtures (Table III), since
dissociation of protein-bound detergent during electrophoresis can lead to protein
aggregation. In contrast, dissociation of digitonin from the lipid/detergent annulus
around membrane proteins seems less important. Therefore, when digitonin is
used for membrane solubilization there is no need to add detergent to the gel.
(2) Electrophoretic mobility of proteins usually is low in CNE, since there is no
anionic dye to shift the charge of solubilized proteins. Therefore, low-percentage
acrylamide gels should be used to assure a certain running distance, for example,
3!13% acrylamide-gradient gels.
The running conditions are the same as described for BNE. At present, no
suitable protocols for electroelution and electroblotting of proteins from CN-gels
are available. However, current investigations promise that CNE can be optimized
to reach the resolution of BNE, and electroblotting of CN-gels can work satisfac-
torily (Wittig and Schagger, manuscript in preparation).
D. Second Dimension: Modified Blue Native Electrophoresis
Two-dimensional native electrophoresis using first dimension CNE and second
dimension BNE has been used to compare the different separation principles of
CNE and BNE (Schagger et al., 1994). In another application, we tested whether
732 Ilka Wittig and Hermann Schagger
supramolecular assemblies of several multiprotein complexes can be dissociated
into the constitutive individual complexes by two-dimensional native electropho-
resis (Schagger and PfeiVer, 2000). We found that BNE containing low levels of
detergent in the cathode buVer B (0.02% dodecylmaltoside or 0.03% Triton X-100)
can dissociate supramolecular structures but retain the structure of the individual
complexes. Since CNE can separate supramolecular protein complexes, we have
used combinations of these native techniques, including CNE/modified BNE
(Fig. 2B) and BNE/modified BNE (not shown).
Procedure
1. For 2D native gels, the first dimension gel should be terminated before
proteins reach their mass-specific pore size limit and stop migrating. This is
necessary to transfer the protein from the first dimension to the second.
2. Strips of 1 cm excised from the first dimension gel are dipped into water for
1 sec, and placed on glass plates at the position usually occupied by the stacking
gel. Spacers (slightly thinner than for first dimension gels, e.g., 1.4 mm) are placed
onto the glass plate. A second glass plate is placed on top of the spacers and held
in place with clamps.
3. Drain excess water before bringing the gel in an upright position.
4. Introduce acrylamide solution that will be used for the second dimension
separation at the gaps between 1D gel strip and spacers, and overlay the acrylam-
ide solution with water.
5. Following polymerization, overlay with more water to cover the 1D native gel
strip, and push the strip on the top of the separating gel using plastic cards. Remove
the water and fill the gaps between gel strip and spacers with a 10% acrylamide
native gel (prepared by analogy with gels in Table III).
6. Add either 0.02% dodecylmaltoside or 0.03% Triton X-100 to cathode buVer B(Table I) and start 2DBNEunder the running conditions for 1DBNE (current limited
to 15 mA; voltage increases gradually during the run from about 200 to 500 V).
7. In contrast to 1D gels, the 2D gels should be terminated late to concentrate
protein into sharp spots. Under these conditions, the intense band of free
Coomassie dye may elute from the gel front.
E. Second or Third Dimension: SDS-PAGE
Although the Tris–Glycine system (Laemmli, 1970) oVers excellent resolution in
1D SDS -PAGE , Tricin e-SDS-P AGE ( Scha gger, 2003a ; Scha gger an d von Jagow ,
1987) is reco mmended for the resol ution of subunit s of complex es in the second
dimension (Fig. 1B). Similarly, Tricine-SDS-PAGE can follow two-dimensional
native electrophoresis (2D CNE/BNE; Fig. 2B) for third dimension resolution
(not shown). Finally, since lower acrylamide concentrations can be used in
Tricine-SDS-PAGE, transfer of proteins to blotting membranes from Tricine
SDS gels is more eYcient than from Tris–Glycine SDS gels.
33. Electrophoretic Methods to Isolate Protein Complexes from Mitochondria 733
Procedure
1. Excise 0.5-cm lanes from 1D native or 2D native gels.
2. Wet gel slices with 1% SDS for 15 min. Solutions containing thiol reagents
are not recommended, except in some rare cases where cleavage of disulfide
bridges is important. In those cases, the strips are wetted for up to 2 h in 1%
SDS, 1% mercaptoethanol, and then briefly rinsed with water, since mercap-
toethanol is an eYcient inhibitor of acrylamide polymerization.
3. Place the equilibrated gel slice on a glass plate at the position usually occupied
by the stacking gel. Apply spacers and place the second glass plate on top of
the spacers. Using spacers that can be thinner that the native gel (e.g., 0.7 mm
for 1.4-mm native gels), 0.5-cm lanes of the native gel are compressed to a width
of about 1 cm and will not move when the glass plates are brought to a vertical
position.
4. Pour the separating gel mixture between the glass plates, usually a 10%
acrylamide gel mixture for Tricine-SDS-PAGE (for the 5- to 100-kDa mass
range and eYcient electroblotting) or a 16% acrylamide gel mixture (for the 5-
to 100-kDa mass range; for sharper bands), until there is a 5-mm gap between the
SDS gel and the native gel strip. Overlay with water to fill the gap.
5. Following polymerization, the native gel strip is gently pushed onto the
separating gel using a 0.6-mm plastic card and residual water is removed.
6. Fill the gaps to the left and right of the native gel strip with a 10% acrylamide
native gel mixture (Table III).
7. Second- or third-dimension SDS-PAGEs using the Tricine-SDS-system and
gel dimensions 0.07�14�14 cm are started at RT, with a maximal voltage of
200 V and current limited to 50 mA. When the current falls below 50 mA, the
voltage can be increased with the current still limited to 50 mA. The run times for
10% and 16% gels are 3–4 h and 5–6 h, respectively.
In some situations, another SDS-gel may follow to complete three- or four-
dimensional electrophoresis (1D native or 2D native electrophoresis are followed
by dSDS-PAGE (the successive application of two orthogonal SDS-gels). dSDS-
PAGE is especially useful when 1D SDS-PAGE is not suYcient to resolve a given
protein mixture properly (Rais et al., 2004). It is also useful when nonreducing and
reducing conditions must be applied sequentially, for example, following use of
chemical cross-linkers containing cleavable disulfide bonds.
III. Applications
There are several principal ways to use native electrophoretic techniques.
It is possible to use 1D native gels for analytical purposes, including in-gel
catalytic activity assays (Wittig and Schagger, 2005; Zerbetto et al., 1997),
colorimetric quantification of protein amounts (Schagger, 1995a, 1996), estimation
734 Ilka Wittig and Hermann Schagger
of native masses and oligomeric states (Schagger et al., 1994), and—following
native electroblotting—for immunological detection and quantification of separated
proteins.
Electroblotting of native gels followed by immunodetection is prone to false
positive detection. For Western blot analysis, subunits of complexes should be
separated by 2D SDS-PAGE, and then identified by specific antibodies (Carrozzo
et al., 2006). Reliability of immunodetection on electroblotted 2D SDS-gels is
much higher, since background signals and cross-reactions can be easily identified
whenever a signal is not detected in the column of protein subunits of the complex
of interest, or the assigned mass is not compatible with the mass of the specific
subunit. Similarly, quantification of protein amounts on Coomassie-stained 2D
SDS-gels is preferable to quantification on 1D BN-gels, since only true subunits of
complexes are used for quantification, whereas protein bands in 1D BN-gels
contain unknown amounts of background protein.
BNE is also used for preparative purposes, applying up to 3-mg protein samples
to a 14-cm wide native gel. Bands that are visible during the run can be excised
and the pro teins can be electroelut ed, as described in Se ction II.B.5. Pr oteins
isolated by preparative BNE have been used for antibody production, protein
characterization, and proteomic studies (Fandino et al., 2005; Rais et al., 2004).
BNE was developed originally as a means to isolate and analyze the five
oxidative phosphorylation complexes from mammalian mitochondria, which are
rather stable membrane protein complexes (Schagger and von Jagow, 1991;
Schagger et al., 2004). It has also been used to analyze protein complexes from
yeast and plant mitochondria (Arnold et al., 1998; Eubel et al., 2003), chloroplasts
(Kugler et al., 1997; Rexroth et al., 2004), and chromaYn granules. Specific
proteins, including vacuolar ATP synthase (Ludwig et al., 1998), bacterial respira-
tory supercomplexes (Stroh et al., 2004), and the mitochondrial protein import
machinery in yeast and plants (Dietmeyer et al., 1997; Jansch et al., 1998), have been
studied using this method. Finally, BNE has been used to study neurotransmitter
assembly (GriVon et al., 1999), apoptosis (Vahsen et al., 2004), and mitochondrial
encephalomyopathies (Bentlage et al., 1995; Carrozzo et al., 2006; Schagger et al.,
2004).
A. Protein Complexes from Mitochondria and Chloroplasts
1. Isolation of Detergent-Stable Mitochondrial Complexes
Protein complexes that can be solubilized by mild nonionic detergents, such as
dodecylmaltoside or Triton X-100, and separated by BNE are defined here as
detergent-stable complexes. The largest detergent-stable multiprotein complexes
that have been isolated from mammalian mitochondria are the oxoglutarate
dehydrogenase complex (OGDC; native mass around 2.5 MDa) and the pyru-
vate dehydrogenase complex (PDC; native mass around 10 MDa), as shown in
ABNE
3.0
3.5
13
Acr
ylam
ide
%
Complex kDa
PDC
OGDCdImIII?
IV
II
10,000
2,5001,4001,000
700490
200
130
966848
30
kDa
10
5
B
BNE PDCOGDC
d III IV III m
SD
S-P
AG
E
Fig. 1 Isolation of detergent-stable mitochondrial complexes by blue native electrophoresis using
Triton X-100 for solubilization. (A) Solubilized bovine heart mitochondrial complexes I–IV (I, II, III,
IV) were separated using a linear 3.5%!13% acrylamide gradient gel for BNE, overlaid by a 3%
acrylamide sample gel. The mitochondrial ATP synthase (also called complex V) was identified as a
majormonomeric form (m) and aminor dimeric form (d).OGDC, oxoglutarate dehydrogenase complex;
PDC, pyruvate dehydrogenase complex. (B) Subunits of the native complexes were separated by Tricine-
SDS-PAGE using a 16.5% T, 3% C gel type. The identity of PDC and OGDC was confirmed by amino-
terminal protein sequencing of the 68-kDa subunit E2 of PDC and the 96- and 48-kDa subunits E1 and
E2, respectively, of OGDC.
33. Electrophoretic Methods to Isolate Protein Complexes from Mitochondria 735
Fig. 1. Concentrations of 3% are the lowest acrylamide concentration that can be
handled routinely in gradient gels. As a result, the size of PDC marks the upper
mass limit of BNE. Prokaryotic ribosomes (around 2–3 MDa) and the larger
eukaryotic ribosomes (6–10 MDa) could be isolated by BNE under special condi-
tions (Schagger, unpublished). However, individual mitochondrial ribosomes (sim-
ilar in size to prokaryotic ribosomes) have not been isolated by BNE, potentially
because they are present in polysomes and are not recovered in the soluble phase
using conventional solubilization conditions.
2. Isolation of Detergent-Labile Supramolecular Assemblies
a. Dimeric and Oligomeric ATP SynthasesThe structural organization of enzymes in biological membranes can be very
complex. An example is ATP synthase, which is usually isolated in monomeric
form. However, electron microscopy revealed that this protein exists as a polymeric
structure that winds as helical double row of particles around tubular cristae of the
mitochondrial inner membrane (Allen et al., 1989).
736 Ilka Wittig and Hermann Schagger
BNE has been used to isolate ATP synthase as dimers or oligomers. This
protein was first isolated as a dimer from yeast mitochondria, using Triton
X-100 at very low detergent/protein ratio and BNE (Arnold et al., 1998). Dimeric
and even oligomeric ATP synthases were later isolated using digitonin from yeast
mitochondria (Paumard et al., 2002; PfeiVer et al., 2003). ATP synthases in the
mass range 1.5–6 MDa have been isolated from mammalian mitochondria (Wittig
and Schagger, 2005). For a discussion of the functional role(s) of oligomeric ATP
synthase, see Wittig et al. (2006).
b. RespirasomesRespirasomes, stoichiometric assemblies of respiratory complexes in the mito-
chondrial membrane, were originally identified when supercomplexes consisting of
complexes I, III, and IV were isolated from bovine heart using BNE (Schagger and
A BNE CNE
S1S0dI
m
III
IV
ht
d
m
III
IV
B
h t d m III First dimension CNE
ImIII
IV
Sec
ond
dim
ensi
on
BN
E (
+D
DM
)
Fig. 2 Analysis of supramolecular assemblies of oxidative phosphorylation complexes using one- and
two-dimensional native electrophoresis techniques. (A) Isolation of supramolecular assemblies of com-
plexes using digitonin for solubilization and blue native electrophoresis (BNE) or clear native electro-
phoresis (CNE) for separation. I, III, and IV, respiratory complexes I, III, and IV, respectively. S0 and
S1, respiratory supercomplexes or respirasomes containing monomeric complex I, dimeric complex III,
and no (0) or one (1) copy of complex IV, respectively. m, d, t, and h, monomeric, dimeric, tetrameric,
and hexameric forms of mitochondrial complex V, respectively. Resolution of BNE is higher compared
to CNE but oligomeric forms of complex V are better preserved in CNE. (B, upper panel) First
dimension CNE (1D CNE) is similar to that in panel A, right lane, but digitonin amounts for solubili-
zation were reduced by 75%. (B, lower panel) Oligomeric forms of complex V were then dissociated into
monomeric complex V (m) during second dimension BNE, which was modified by adding dodecylmal-
toside [2D BNE (þDDM)].
33. Electrophoretic Methods to Isolate Protein Complexes from Mitochondria 737
PfeiVer, 2000) (Fig. 2). Moreover, analyses of muscle tissue and cell lines of
patients with mitochondrial disorders indicated that association of respiratory
complexes into respirasomes is essential for complex I stability (Acin-Perez
et al., 2004; Schagger et al., 2004). Assembly of respiratory complexes into respira-
somes may also facilitate channeling of the electron carrier quinone between com-
plexes I and III, which makes electron transfer rates essentially independent of the
midpoint potential of the quinone pool (Bianchi et al., 2004). Interestingly, recent
evidence suggested that respirasomes are not the largest assemblies of respiratory
complexes in themembrane. Specifically, electronmicroscopy studies have revealed
that large particles are arranged at regular intervals on mitochondrial inner
membranes (Allen, 1995; Allen et al., 1989). Therefore, it is possible that respira-
somes, with masses up to 2.3 MDa in mammalian mitochondria, associate in a
larger linear structure, which we refer to as the ‘‘respiratory string’’ (Wittig et al.,
2006).
3. Determination of Molecular Masses of Native Protein Complexes
As shown in Fig. 1, using dodecylmaltoside or Triton X-100 for solubilization
BNE can separate proteins according to their molecular masses. Many commer-
cially available water-soluble proteins and the bovine mitochondrial complexes
have been shown to fit a general calibration curve (Schagger, 2003b; Schagger
et al., 1994). With the exception of some extremely basic membrane proteins and
some water-soluble proteins that do not bind Coomassie dye, the native masses of
membrane proteins can be determined with a maximal error of 20%.
Using digitonin instead of dodecylmaltoside or Triton X-100 for BNE, the
migration of membrane protein in general is slightly reduced. Since digitonin
delipidates membrane proteins less than does dodecylmaltoside or Triton X-100,
the lower migration of complexes solubilized in digitonin compared to other
detergents may be due to altered protein/lipid/detergent/Coomassie ratios within
solubilized complexes. Therefore, for mass determination using BNE, solubilize
membrane proteins in the same detergent as the protein of interest. Estimation
of native masses in CN-gels is more complicated, as described by Wittig and
Schagger (2005).
4. In-Gel Catalytic Activity Measurements
Coomassie dye can adversely aVect catalytic activity assays (Schagger and von
Jagow, 1991). In spite of this, Zerbetto et al. (1997) have measured activity of
oxidative phosphorylation complexes separated in BN-gels. Not surprisingly, the
same assays can be used for CN-gels. In CN-gels, the assays can be much faster
and inhibitor sensitive (PfeiVer et al., 2003; Wittig and Schagger, 2005).
738 Ilka Wittig and Hermann Schagger
B. Isolation of Protein Complexes from Tissue Homogenates and Cell Lines
Mitochondrial protein represents about 1%, 5%, and 10% of the total cellular
protein in cell lines, skeletal muscle, and heart muscle, respectively. This corre-
sponds to less than 10 mg total mitochondrial protein in 10 mg of sedimented cells
(wet weight), or less than 100 mg in 10-mg heart tissue. The lower protein amounts
were suYcient for in-gel activity assays and silver staining. The larger protein
amounts even allowed for Coomassie staining of 2D gels and densitometric
quantification of the subunits of respiratory complexes. Protocols for processing
diVerent tissues (Bentlage et al., 1995; Schagger and Ohm, 1995) and cell lines
(Schagger et al., 1996) have been described elsewhere, and will not be repeated
here.
C. Purification of Partially Purified Protein Complexes Using BN-PAGE
Purification of membrane proteins and complexes is often diYcult, and only
partial purification may be achieved by conventional isolation protocols. BNE is a
valuable means for final purification of these problematic proteins and has been
used as a final purification step for antibody production and protein chemistry
studies. One example of this type of purification is described in Schagger (1995b).
About 1–2 mg of a partially purified mitochondrial complex could be loaded on
each preparative gel and recovered by native electroelution. Three general rules
may help to avoid dissociation of prepurified complexes during final purification.
1. The salt concentration in the sample should be low, but suYcient to keep
proteins in solution, for example, 25- to 50-mM NaCl. High salt causes protein
concentration and aggregation within the sample well during electrophoresis.
2. Keep detergent concentrations for protein elution from chromatographic
columns low (e.g., 0.05% Triton X-100 or 0.1% dodecylmaltoside), because the
combination of a neutral detergent and the anionic Coomassie dye used subse-
quently in BNE exhibits some properties of a mild anionic detergent.
3. Do not add Coomassie dye to the sample if the desired protein complex
contains detergent-labile subunits. The Coomassie dye that is required in BNE is
supplied with cathode buVer B. Cathode buVer B/10 can be used directly when the
detergent concentration in the sample is as low as 0.2%.
IV. Outlook
BNE and CNE preserve some physiological protein–protein interactions, and
allow for the isolation of special supramolecular protein assemblies. However,
weak and dynamic interactions in a cell or organelle may not be detected using this
method. Chemical cross-linking often is required to fix two interacting proteins
together. Multidimensional electrophoresis, using native conditions first, and
33. Electrophoretic Methods to Isolate Protein Complexes from Mitochondria 739
dSDS-PAGE last, can then be used to prepare samples for the mass spectrometric
identification of the physiologically associated proteins. This application may
become an essential alternative to two-hybrid systems, or an experimental assay
to verify or dismiss predicted interactions by two-hybrid systems.
Acknowledgment
This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 628,
Project P13 (H.S.).
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