[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-PAGE
III. 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-PAGE
IV. O
utlook R eferences
I. 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.


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.


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.


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.


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


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


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


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.


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


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.


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).


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)].


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).


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


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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