Biochemistry

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for the Identification and Analysis of Multiprotein Complexes

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Science's STKE  25 Jul 2006:
Vol. 2006, Issue 345, pp. pl4
DOI: 10.1126/stke.3452006pl4

Abstract

Multiprotein complexes (MPCs) play crucial roles in cell signaling. Two kinds of MPCs can be distinguished: (i) Constitutive, abundant MPCs--for example, multisubunit receptors or transcription factors; and (ii) signal-induced, transient, low copy number MPCs--for example, complexes that form upon binding of Src-homology 2 (SH2) domain-containing proteins to tyrosine-phosphorylated proteins. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a separation method with a higher resolution than gel filtration or sucrose density ultracentrifugation that can be used to analyze abundant, stable MPCs from 10 kD to 10 MD. In contrast to immunoprecipitation and two-hybrid approaches, it allows the determination of the size, the relative abundance, and the subunit composition of an MPC. In addition, it shows how many different complexes exist that share a common subunit, whether free monomeric forms of individual subunits exist, and whether these parameters change upon cell stimulation. Here, we give a detailed protocol for the separation of MPCs from total cellular lysates or of prepurified MPCs by one-dimensional BN-PAGE or by two-dimensional BN-PAGE and SDS-PAGE.

Introduction

Protocol Video

This video, a joint project of Science Signaling and the Journal of Visualized Experiments, demonstrates the steps outlined in this protocol article.

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Most, if not all, proteins require binding to other proteins to function in a regulated manner. These regulatory and functional interactions result in the formation of multiprotein complexes (MPCs). Examples range from the transcription factor nuclear factor-κB (NF-κB), which is inhibited by binding to its inhibitor IκB; to the tyrosine kinase Syk, which is activated by binding to doubly phosphorylated immunotyrosine activation motifs (ITAMs); and to phosphoinositide 3-kinase (PI3K), the localization of which changes upon association with receptor tyrosine kinases. Most proteins may be part of several distinct MPCs, as well as being present as monomers. The abundance of distinct MPCs of which a certain protein is a part can vary enormously. In addition, MPCs may exhibit spatial and temporal changes, as well as exhibit different stabilities. Therefore, identifying and analyzing MPCs is a difficult task.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) has advantages for the study of MPCs in that it can provide information about the size, number, protein composition, stoichiometry, or relative abundance of MPCs. For example, if proteins A and B copurify with protein X, BN-PAGE can often distinguish an A-B-X complex from two independent A-X and B-X complexes (1, 2). We have modified the original BN-PAGE protocol of H. Schägger for general applicability (3) and used it to study signaling proteins (36).

The dye Coomassie blue, which binds nonspecifically to all proteins and is itself negatively charged, is used in BN-PAGE. Therefore, the electrophoretic mobility of an MPC is determined by the negative charge of the bound Coomassie blue dye and the size and shape of the complex (Fig. 1) (1, 2). Coomassie blue does not act as a detergent, and it preserves the structure of MPCs. In contrast to other native gel electrophoresis systems, MPCs are separated independently of their isoelectric point and, therefore, the size of the MPCs can be estimated (1, 79). In addition, the binding of Coomassie blue to proteins decreases their tendency to aggregate during the stacking step of the electrophoresis process.

Fig. 1.

Principle of BN-PAGE. Proteins and MPCs are separated under native conditions in a first-dimension BN-PAGE. For a two-dimensional BN-PAGE and SDS-PAGE, the proteins and/or MPCs are denatured by SDS in the gel strip after they are separated by BN-PAGE, then applied to a second-dimension SDS-PAGE gel. The hyperbolic shape of the diagonal in the two-dimensional gel is due to a gradient gel in the first and a linear gel in the second dimension. Monomeric proteins are located on the diagonal and the components of MPCs below the diagonal. See also Fig. 6. [Reprinted from Molecular and Cellular Proteomics, Vol. 3, M. M. Camacho-Carvajal et al., "Two-dimensional blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates," pp. 176–182, copyright (2004), with permission from the American Society for Biochemistry and Molecular Biology]

Abundant, stable MPCs can easily be studied by BN-PAGE. These are, for example, complexes that are constitutively present in a cell, independent of receptor stimulation. BN-PAGE has been used to identify the complex composed of the adaptors Gads and SLP-76 (4); B cell receptor (BCR) and T cell receptor (TCR) antigen receptor complexes (5, 6, 1013); complexes of transcription factors (3); and the phosphatase CD45 (5). However, receptor stimulation-induced complexes, such as those created through the binding of Src-homology 2 (SH2) domain-containing proteins to tyrosine-phosphorylated proteins, could not be resolved, which may be due to the low abundance of these transient MPCs (4). We suggest methods for enriching samples for these transient MPCs, thereby extending this technique to complexes induced during cell signaling.

Although the conclusions that can be drawn from coimmunoprecipitation experiments are limited, these experiments can be a valuable starting point for MPC analysis. Therefore, before attempting to analyze MPCs using BN-PAGE, we recommend testing whether the protein complex of interest can be detected using coimmunoprecipitation experiments. Successful copurification indicates that the MPC of interest is stable and abundant enough to be detected by BN-PAGE. For many MPCs, solubilization of the complex requires the use of detergents. It is best to compare several detergents of different classes in preliminary experiments, including detergents that allow successful coimmunopurifications of the proteins expected to be present in a common MPC. Three technical steps that are crucial for performing high-resolution BN-PAGE include: (i) preparation of the sample with low ionic strength (3); (ii) selection of an appropriate solubilization detergent (5, 14); and (iii) performing the electrophoresis. The best detergent must be selected empirically and can vary from one protein complex to another. Precast BN-gels are commercially available and should facilitate the application of this technique.

Materials

6-aminohexanoic acid (ε-aminocaproic acid)

Note: This chemical is an irritant and should be handled with gloves.

30% H2O2

Acrylamide-bisacrylamide solutions, 19:1 and 37.5:1 [Rotiphorese Gel 40 (Roth, Karlsruhe, Germany)]

Note: This solution is neurotoxic and should be handled with gloves.

Antibody that recognizes the protein of interest in Western blotting

Ammonium persulfate (APS)

Beads coupled to antibodies against phosphotyrosine residues [for example, the antibody 4G10 coupled to agarose (Upstate Biotech, Dundee, UK)]

Bis-tris

Bromophenol blue

β-mercaptoethanol

Coomassie blue G250 (Serva, Heidelberg, Germany)

Note: Do not substitute other types of Coomassie dye such as Coomassie blue R250 or colloidal Coomassie blues.

Dialysis membranes, molecular weight cut-off 10 to 50 kD

Glycerol

Isopropanol

N,N,N′,N′-Tetramethylethylenediamine (TEMED)

Parafilm

Precast BN-gels and buffers [optional, available from Invitrogen (NativePAGE Novex Bis-Tris Gel System)]

Phenylphosphate

Polyvinylidene difluoride (PVDF) membrane (Immobilon P, Millipore)

Sodium dodecyl sulfate (SDS)

Tris-HCl

Tricine

Silver stain kit (for example, Bio-Rad or Pierce)

Detergents

Brij 96 (Sigma-Aldrich, Taufkirchen, Germany)

Digitonin (Sigma-Aldrich)

Dodecylmaltoside (Applichem, Darmstadt, Germany)

Triton X-100 (Roth)

Note: Digitonin and Triton X-100 are toxic, and gloves should be worn when handling buffers or samples containing these detergents. The best detergent to solubilize, but not disrupt, the MPC must be empirically determined. Other detergents than those listed can also be tested.

Protease and Phosphatase Inhibitors

Note: These inhibitors are toxic and should be handled with gloves.

Aprotinin

Leupeptin

Phenyl methyl sulfonyl fluoride (PMSF)

Sodium fluoride

Sodium orthovanadate

Marker Proteins

Aldolase (Sigma-Aldrich)

Bovine serum albumin (BSA) (Sigma-Aldrich)

Catalase (Sigma-Aldrich)

Ferritin (Sigma-Aldrich)

Thyroglobulin (Sigma-Aldrich)

Equipment

Centrifuges

Cold room or large refrigerator

Gel electrophoresis system [minigel (Bio-Rad Protean II or III) or large gel (Bio-Rad Protean XI)]

Note: Other suppliers include Hoefer and Pharmacia Biotech.

Gradient mixer (self-made or commercially available from Bio-Rad laboratories)

Magnetic stirrer

Peristaltic pump

Power supply

Rotating wheel

Semi-dry transfer equipment (for example from Bio-Rad)

Silicon tubing, 3 to 5 mm diameter, 1 m length (NeoLab, Heidelberg, Germany)

Recipes

Note: Prepare all buffers with distilled H2O (dH2O).

Recipe 1: Phosphate-Buffered Saline (PBS)
Na2HPO48.1 mM
KH2PO41.5 mM
NaCl138 mM
KCl2.7 mM
Prepare 1 liter. Solution should be pH 7.4 if prepared properly.
Recipe 2: Protease and Phosphatase Inhibitor Stock Solutions
Leupeptin (1000x): Dissolve 10 mg/ml in dH2O. Store in 1-ml aliquots at 20°C.
Aprotinin (1000x): Dissolve 10-mg/ml solution in dH2O. Store in 1-ml aliquots at 20°C.
PMSF (100x): Prepare a 100-mM solution in ethanol. Store in 1-ml aliquots at 20°C.
Sodium orthovanadate (100x): Prepare a 50-mM solution in dH2O. Store at room temperature.
Sodium fluoride (100x): Prepare a 50-mM solution in dH2O. Store at room temperature.
Recipe 3: BN-Lysis Buffer
Base buffer
Bis-tris20 mM
ε-aminocaproic acid500 mM
NaCl20 mM
EDTA, pH 8.02 mM
Glycerol10%
Adjust pH of buffer to 7.0 with HCl. Store at 4°C.
Detergent
Digitonin0.5 to 1.0%
or
Triton X-1000.1 to 0.5%
or
Brij 960.1 to 0.5%
or
Dodecylmaltoside0.1 to 0.5%
Protease and phosphatase inhibitors
Aprotinin (Recipe 2)10 μg/ml
Leupeptin (Recipe 2)10 μg/ml
PMSF (Recipe 2)1 mM
Sodium fluoride (Recipe 2)0.5 mM
Sodium orthovanadate (Recipe 2)0.5 mM
5 to 10 ml of BN-Lysis Buffer is sufficient for 10 samples.
Note: The appropriate detergent(s) must be determined empirically and should be the same as that used in the other lysis buffer recipes. Digitonin must be added just before use from a 2% stock solution in dH2O (store in 5-ml aliquots at −20°C). Protease and phophatase inhibitors should be added immediately before use. Upon addition of orthovanadate, the buffer will become yellowish in color.
Recipe 4: BN-Dialysis Buffer
Base buffer
Bis-tris20 mM
ε-aminocaproic acid500 mM
NaCl20 mM
EDTA, pH 8.02 mM
Glycerol10%
Adjust pH of buffer to 7.0 with HCl. Store at 4°C.
Detergent
Digitonin0.3 to 0.5%
or
Triton X-1000.1%
or
Brij 960.1%
or
Dodecylmaltoside0.1%
Protease and phosphatase inhibitors
PMSF (Recipe 2) 1 mM
Sodium orthovanadate (Recipe 2)0.5 mM
500 ml of BN-Dialysis Buffer is sufficient for 10 samples.
Note: The appropriate detergent(s) must be determined empirically and should be the same as that used in the other lysis buffers, but at the indicated lower concentrations. Detergent must be added to prevent aggregation at the stacking step of gel electrophoresis. Protease and phophatase inhibitors should be added immediately before use.
Recipe 5: Lysis Buffer
Base buffer
Tris-HCl20 mM
NaCl137 mM
EDTA, pH 8.02 mM
Glycerol10%
Adjust pH of buffer to 7.4 with HCl. Store at 4°C.
Detergent
Digitonin0.5 to 1.0%
or
Triton X-1000.1 to 0.5%
or
Brij 960.1 to 0.5%
or
Dodecylmaltoside0.1 to 0.5%
Protease and phosphatase inhibitors
Aprotinin (Recipe 2)10 μg/ml
Leupeptin (Recipe 2)10 μg/ml
PMSF (Recipe 2)1 mM
Sodium fluoride (Recipe 2)0.5 mM
Sodium orthovanadate (Recipe 2)0.5 mM
5 ml of Lysis Buffer is sufficient for 10 samples.
Note: The appropriate detergent(s) must be determined empirically and should be the same as that used in the other lysis buffer recipes. Digitonin must be added just before use from a 2% stock solution in dH2O (store in 5-ml aliquots at −20°C). Protease and phophatase inhibitors should be added immediately before use.
Recipe 6: BN-Dialysis Buffer Containing Phenylphosphate
Add 100 mM phenylphosphate to BN-Dialysis Buffer (Recipe 4).
Recipe 7: 3x BN-Gel Buffer
Bis-tris150 mM
ε-aminocaproic acid200 mM
Adjust pH to 7.0 with HCl. Store at 4°C. 15 ml of BN-Gel Buffer are sufficient for one 30-ml gel.
Recipe 8: Acrylamide-Bisacrylamide Mix
Mix 17.3 ml of 19:1 acrylamide-bisacrylamide with 82.7 ml of 37.5:1 acrylamide-bisacrylamide. This will result in a ratio of 32:1 with 40% acrylamide. Store at 4°C.
Recipe 9: 4% Separating Gel
3x BN-Gel Buffer (Recipe 7)5.00 ml
Acrylamide-Bisacrylamide Mix (Recipe 8)1.50 ml
dH2O8.50 ml
APS, 10% in dH2O54 μl
TEMED 5.4 μl
Note: Add APS and TEMED immediately before pouring gel, as these reagents promote polymerization. This recipe is sufficient to cast a 30-ml gel. Adjust volumes appropriately for the number and size of the gels being poured.
Recipe 10: 15% Separating Gel
3x BN-Gel Buffer (Recipe 7)5.00 ml
Acrylamide-Bisacrylamide Mix (Recipe 8)5.63 ml
Glycerol 70%4.38 ml
APS, 10% in dH2O42 μl
TEMED4.2 μl
Note: Add APS and TEMED immediately before pouring gel, as these reagents promote polymerization. This recipe is sufficient to cast a 30-ml gel. Adjust volumes appropriately for the number and size of the gels being poured. The concentration of acrylamide-bisacrylamide may also be varied as necessary from 10 to 18%. This recipe results in a 4 to 15% gel.
Recipe 11: 3.2% Stacking Gel
3× BN-gel Buffer (Recipe 7)3.00 ml
Acrylamide-Bisacrylamide Mix (Recipe 8)0.72 ml
dH2O5.28 ml
APS, 10% in dH2O120 μl
TEMED12 μl
Note: Add APS and TEMED immediately before pouring gel, as these reagents promote polymerization. This recipe is sufficient to cast a 30-ml gel. Adjust volumes appropriately for the number and size of the gels being poured.
Recipe 12: Cathode Buffer
Bis-tris15 mM
Tricine50 mM
Coomassie blue G250 0.02%
Prepare 1 liter as a 10x stock, adjust pH to 7.0 with HCl, and store at 4°C. Dilute 1:10 with dH2O before use.
Recipe 13: 100x Pervanadate
Sodium orthovanadate (Recipe 2)5 μl
dH2O5.7 μl
H2O2, 30%1.5 μl
Mix in the order listed and then incubate 5 to 30 min at room temperature until solution becomes brownish.
Recipe 14: Marker Mix
Aldolase (158 kD)10 mg/ml
Catalase (232 kD)10 mg/ml
Ferritin (440 and 880 kD)10 mg/ml
Thyroglobulin (670 kD)10 mg/ml
BSA (66 and 132 kD)10 mg/ml
Bis-tris20 mM
NaCl 20 mM
Glycerol 10%
Adjust pH to 7.0 with HCl. Store at 4°C.
Note: Molecular weight markers are also commercially available from several sources, including Invitrogen or Pharmacia.
Recipe 15: Anode Buffer
Bis-tris50 mM
Prepare 1 liter as a 10× stock, adjust pH to 7.0 with HCl, and store at 4°C. Dilute 1:10 with dH2O before use.

Recipe 16: SDS Sample Buffer
Amount Reagent Concentration
0.151 gTris12.5 mM
4 gSDS4%
20 gGlycerol20%
20 mgBromophenol blue 0.02%
Adjust volume to 100 ml with dH2O. Adjust pH to 6.8. Store at room temperature.
Note: To reduce disulfide bonds, add 9 ml β-mercaptoethanol. SDS as a powder and β-mercaptoethanol are toxic; therefore, use gloves and work under a hood.
Recipe 17: Semidry Transfer Buffer
Amount Reagent Concentration
5.81 gTris48 mM
2.93 gGlycine39 mM
200 mlMethanol20%
1 gSDS0.1%
Adjust volume to 1 liter with dH2O. Store at room temperature.
Recipe 18: Wet Transfer Buffer
Amount Reagent Concentration
3 gTris25 mM
14.4 gGlycine190 mM
200 mlMethanol20%
1 gSDS0.1%
Adjust volume to 1 liter with dH2O. Store at room temperature.
Recipe 19: Destaining Solution
Prepare a 45% methanol, 10% acetic acid solution. Store at room temperature.

Instructions

Sample Preparation Methods

Samples for BN-PAGE may be prepared from several sources. To increase the likelihood that a MPC will be detectable, in addition to the basic preparation methods from total cell lysates, we provide two alternative sample preparation methods that allow enrichment of samples for complexes with phosphotyrosine-containing proteins or with membrane proteins. In addition, more specific affinity-purification protocols may be used that allow native elution of the proteins from the affinity matrix—for example, the tandem affinity purification (TAP-tag) method (15).

Before preparing the samples for BN-PAGE, the optimal detergent and concentration that will preserve the MPCs yet solubilize the cells should be determined. Commonly used detergents that can be tested include digitonin (0.5 to 1%), Triton X-100 (0.1 to 0.5%), Brij 96 (0.1 to 0.5%), or dodecylmaltoside (0.1 to 0.5%). These reagents are nonionic detergents, which tend to be best for MPC stability. Other detergents can also be used [information about detergents can be found online; see (16)]. Even if soluble MPCs are to be analyzed, detergents must be present in the dialysis step to prevent aggregation during the stacking step of the gel run.

The first sample preparation instructions describe how to prepare total cell lysates. The details of cell culture will be specific to the cells used for the experiment and are therefore not included here. Lysates may also be prepared from tissue samples.

Preparation of total cell lysates

1. Harvest 2 x 106 cells and pellet by centrifugation at 350g for 5 min at 4°C.

Note: Suspension cells can be harvested by centrifugation; cells that grow attached to the culture dishes should be released from the dishes with 0.5 mM EDTA (avoid trypsin, because it digests extracellular proteins); and tissues should be homogenized with a Dounce homogenizer in PBS (Recipe 1).

2. Wash the cell pellet three times with 0.5 ml of ice-cold PBS (Recipe 1), and pellet after each wash by centrifugation at 350g for 5 min at 4°C.

3. Resuspend the cell pellet at 2 x 106 cells per 100 μl of ice-cold BN-Lysis Buffer (Recipe 3).

4. Incubate on ice for 15 min.

5. Centrifuge at 13,000g for 15 min at 4°C.

6. Melt a hole in the cap of a microcentrifuge tube using a hot Pasteur pipette (Fig. 2A), and then place the tube on ice to chill.

Fig. 2.

Dialysis of the total cell lysate. (A) For each sample, a hole is made in the cap of a microcentrifuge tube using a hot Pasteur pipette. (B) After cooling the tube to 4°C and addition of the sample, a dialysis membrane (molecular weight cut-off of 10 kD) is placed on top of the tube. (C) The cap is closed, any excess dialysis membrane that sticks out is trimmed off, and the ring between tube and cap is sealed with Parafilm. (D) After a short upside-down centrifugation of the tube, the tube is placed and affixed with tape inside a beaker containing the dialysis buffer and a magnetic stirrer. The air bubbles at the dialysis membrane are removed with a bent Pasteur pipette. Care is taken not to damage the membrane. Dialysis is performed at 4°C for at least 6 hours with continuous stirring.

7. Transfer the supernatant from step 5 into the chilled tube with the hole in the cap.

8. Place a dialysis membrane with forceps over the opened tube and close the cap (F2, B and C).

Note: Ensure that there are no folds or tears in the dialysis membrane.

9. Seal the cap carefully with Parafilm.

10. Invert the tube and centrifuge upside-down at the lowest speed possible in the adaptor cavity for 50-ml conical tubes in a cell culture centrifuge for 10 s at 4°C.

Note: Remove the inverted tube from the centrifuge using large tweezers to avoid turning the tube right side up.

11. Prepare a 100 ml beaker with cold BN-Dialysis Buffer (Recipe 4) and a magnetic stirrer. Use at least 10 ml of BN-Dialysis Buffer per 100-μl sample.

12. Affix the tube with tape upside-down inside the beaker, and remove air bubbles from the hole beneath the cap using a bent Pasteur pipette (Fig. 2D).

13. Switch on the magnet stirrer and leave it for 6 hours or overnight in the cold room. Check occasionally to ensure that stirring is not creating air bubbles at the dialysis membrane.

14. Collect the dialyzed cell lysate in a new chilled microcentrifuge tube.

Purification of phosphotyrosine-containing proteins for BN-PAGE

It is possible to enrich for the MPCs of interest; however, commonly used immunoprecipitation methods cannot be combined with BN-PAGE, because it is not possible to elute the MPC under native conditions from the antibody-coupled beads. MPCs with components that are phosphorylated on tyrosine residues may be immunopurified using antibodies against phosphotyrosine. The MPCs are then eluted with an excess of phenylphosphate, which competes with phosphotyrosine for binding to the antibody. Here, we provide detailed instructions for preparation of phosphotyrosine-enriched samples.

1. Harvest 2 x 107 cells and pellet by centrifugation at 350g for 5 min at 4°C.

Note: Cells may be stimulated with ligands, agonists, antagonists, or other conditions, depending on the experiment.

2. Wash the cell pellet once with 1 ml of ice-cold PBS (Recipe 1) and pellet by centrifugation at 350g for 5 min at 4°C.

3. Resuspend the cell pellet at 2 x 107 cells per 1 μl of ice-cold Lysis Buffer (Recipe 5).

4. Incubate for 15 min on ice.

5. Centrifuge at 13,000g for 15 min at 4°C.

6. Transfer the supernatant from step 5 into a new chilled tube.

7. Add 10 μl of beads coupled to antibodies against phosphotyrosine residues and incubate on a rotating wheel for 2 hours to overnight in the cold room.

8. Centrifuge at 400g for 2 min at 4°C.

9. Wash the beads three times with 1 ml of ice-cold BN-Dialysis Buffer (Recipe 4), centrifuging 400g for 2 min at 4°C.

10. Add 40 μl of ice-cold BN-Dialysis Buffer Containing Phenylphosphate (Recipe 6) and resuspend the beads using a 20-μl pipette tip that has been cut at the narrow end to make the opening larger.

11. Resuspend beads every 5 min for 30 min while incubating on ice.

Note: If desired, proteins may be dephosphorylated by adding one unit of alkaline phosphatase to the eluate during the last 5 min of the elution procedure.

12. Centrifuge at 400g for 2 min at 4°C and collect the supernatant (eluate) in a new, chilled tube.

Note: These samples do not have to be dialyzed and are ready for BN-PAGE.

Preparation of membranes for BN-PAGE

MPCs that are associated with cell membranes or inside organelles can be enriched before preparing the lysate for analysis by BN-PAGE.

1. Prepare membrane fractions of cells using standard protocols (17).

2. Wash the membrane pellet once with 0.5 ml of ice-cold BN-Lysis Buffer (Recipe 3) without detergent.

3. Resuspend the membrane pellet completely without generating air bubbles in ice-cold BN-Lysis Buffer (Recipe 3), including detergent. Use the equivalent of 4 x 107 cells per 100 μl of BN-Lysis Buffer.

4. Incubate for 1 hour at 4°C, resuspending the pellet every 15 min.

5. Centrifuge at 20,000g for 10 min at 4°C.

6. Collect the supernatant (membrane lysate) in a new, chilled microcentrifuge tube.

Note: These samples do not have to be dialyzed and are ready for BN-PAGE.

Pouring the BN-Gel

Gradient gel pouring is done at room temperature with a gradient mixer (Fig. 3). Because of its high glycerol content, the gel mix with the higher-percentage (15%) acrylamide-bisacrylamide is heavier than the low-percentage (4%) gel. This density difference aids in establishment of a uniform gradient between the glass plates. Gloves must be worn, because polyacrylamide is highly neurotoxic. Note that precast BN-gels and buffers are commercially available from Invitrogen (NativePAGE Novex Bis-Tris Gel System).

Fig. 3.

Pouring the BN-gradient gel. The gradient mixer contains two cylinders into which the low- (4%) and high- (15%) percentage separating gel solutions are added (indicated as "low" and "high" at the upper left of the photograph). These cylinders are connected by a channel that can be closed and opened by a valve. The magnetic stirrer is placed into the cylinder containing the high-percentage gel mix. The outflowing flexible tube can be closed by a clamp. After passing the peristaltic pump, the tube ends with a syringe needle, which is placed into the top between the two glass plates of the gel equipment.

1. Place the gradient maker on a stir plate and attach to a peristaltic pump as shown in Fig. 3. Close the channel using the valve and close the tubing with a clamp.

2. Attach a syringe needle to the end of a piece of flexible tubing that comes out of the peristaltic pump and place the needle into the top, between the two glass plates of the gel apparatus. Place the needle close to the bottom and raise it slowly as the gel pours.

3. Prepare 4% and 15% Separating Gels (Recipes 9 and 10), adding APS and TEMED only immediately before use.

Note: The volumes of the two gel solutions combined should be exactly equal to the volume required to fill the space between the glass plates to the required height.

4. Pour these gel solutions into the corresponding cylinders of the gradient mixer (4% into the "low" and 15% into the "high" cylinder) (Fig. 3).

5. Open the valve and force out the air bubble inside the channel connecting the two gel reservoirs by pressing over the left cylinder with your thumb.

6. Switch on the pump to 5 ml per minute, remove the clamp, and allow the gel to slowly flow between the glass plates. Ensure that the needle is always above the height of the liquid, so that the gradient is not disturbed.

7. Allow all liquid to enter the gel apparatus, and then overlay gently with isopropanol. Allow the gel to polymerize for at least 30 min at room temperature.

8. Clean the pouring apparatus with dH2O (do not use detergent).

9. Remove the isopropanol, wash with dH2O, and remove the dH2O carefully with a slip of absorbent paper without touching the gel.

10. Prepare a 3.2% Stacking Gel (Recipe 11), adding APS and TEMED only immediately before use.

11. Pour the stacking gel on top of the separating gel and immediately introduce the comb between the glass plates, avoiding bubbles at the interface between the gel solution and the comb. Allow the gel to polymerize.

Note: Make sure that at least 0.5 cm (for 5-ml minigels) or 2.5 cm (for large, 30- to 50-ml gels) of stacking gel remain between the comb and the separating gel.

12. Remove the comb slowly, pulling it out at an angle to the plane of the gel. This allows air to enter the pockets rapidly, which improves the quality of the wells.

Separation of the Sample by BN-PAGE

Coomassie blue is present in the solution at the cathode that is overlayed onto the samples that have been added to the wells, and it interacts with the MPCs inside the wells of the gel and enters the gel during electrophoresis (Fig. 4), preventing aggregation of proteins in the stacking gel. Once the samples are prepared, the remainder of this part of the procedure should be performed at 4°C. We recommend boiling an aliquot of the sample with SDS to disrupt the MPCs as a control and loading it also on the BN-PAGE.

Fig. 4.

Electrophoresis using BN-PAGE. (A) First, the samples are added to the pockets of the pre-cooled gel and overlaid with the Cathode Buffer (Recipe 12). Second, the gel is placed into the electrophoresis equipment, the inner chamber is filled with the Cathode Buffer (Recipe 12), and the outer chamber is filled with the Anode Buffer (Recipe 15). Third, the electric field is applied to start the electrophoresis. The marker protein ferritin can be seen by its brownish color (arrow). (B) After electrophoresis, the running front is seen as a dark blue line (vertical arrow), whereas the rest of the gel is light blue because the Coomassie blue entered it. The marker protein ferritin can be seen by its brownish color (tilted arrow).

1. Boil an aliquot of the sample, to be used as a control, in 1% SDS for 5 min to dissociate all MPCs. Leave one lane empty between this control and the "non-SDS samples."

2. Load 1 to 20 μl of sample in the dry wells, before adding the Cathode Buffer (Recipe 12).

Note: If the sample contains phosphorylated proteins and the phosphorylation is to be preserved, add 1/100th volume of 100x Pervanadate (Recipe 13) to the dialyzed lysate.

3. Load 10 μl of Marker Mix (Recipe 14).

Note: Only ferritin is visible during the electrophoresis due to its brown color ( Fig. 4A , arrow). The other markers will be visible following Coomassie or silver staining.

4. Overlay the samples in each well with Cathode Buffer (Recipe 12) (Fig. 4A).

5. Fill the inner chamber with Cathode Buffer (Recipe 12) and the outer and lower chambers with Anode Buffer (Recipe 15).

6. Apply voltage to a minigel at 100 V or a large gel at 150 V, until the samples have entered the separating gel.

7. Increase the voltage to 180 V (minigel) or 400 V (large gel) and run until the dye front reaches the end of the gel.

Note: The gel run takes between 3 and 4 hours for a mini-gel, and between 18 and 24 hours for a large gel.

Second-Dimension SDS-PAGE

After performing the first-dimension BN-PAGE, it is possible to run a second-dimension SDS-PAGE to separate each MPC into its components.

1. Prepare a standard SDS-PAGE gel (17) with a single large lane and one lane for molecular weight markers.

Note: To make it easier to load the BN-PAGE gel slice onto the second-dimension gel, we tape (Tesafilm or Cellotape) the spacers and comb of the SDS-PAGE gel. This results in a slightly thicker gel, which is sufficient to allow the BN-PAGE gel slice to slip between the plates and into the well easily, but still remain fixed between the glass plates.

2. Remove the BN-PAGE gel in the plates from the electrophoresis apparatus and gently pry up one plate.

3. Cut out the lane of the BN-PAGE gel containing the proteins of interest and remove the stacking gel.

4. Place the BN-PAGE gel slice in SDS Sample Buffer (Recipe 16) (5 ml for a minigel slice) in a small dish or cell culture plate, and incubate for 10 min.

5. Boil the BN-PAGE gel slice briefly (not more than 20 s) in the microwave.

Note: Excessive boiling will cause the gel slice to shrink.

6. Continue incubating the BN-PAGE gel slice in the hot SDS Sample Buffer (Recipe 16) for another 15 to 20 min.

7. Load the BN-PAGE gel slice over the stacking gel of the SDS-PAGE gel, and overlay the slice with SDS Sample Buffer (Recipe 16).

8. Perform electrophoresis according to standard protocols.

Visualization of MPCs

Several methods are available for visualizing the protein complexes. The protein constituents of MPCs that have been isolated by BN-PAGE or second-dimension SDS-PAGE can be analyzed by Coomassie blue staining or silver staining. Coomassie blue is a good choice for highly abundant MPCs (μg amounts), whereas silver staining is good for visualization of 50 to 1000 ng amounts of the protein of interest. These methods are standard and the details are not provided here.

Another option is Western blotting. The advantages of Western blotting are that it is a sensitive method and the presence of specific proteins can be determined. However, some antibodies do not react properly after a first-dimension BN-PAGE. In these cases, the sample must be subjected to second-dimension SDS-PAGE. To detect all subunits of a MPC, Western blotting requires antibodies to each subunit if the MPC has been separated into its constituents by second-dimension SDS-PAGE. Alternatively, the proteins in the MPC must be epitope-tagged so that they can be identified with commercially available antibodies. Transfer of the proteins from a BN-PAGE gel to a membrane (nitrocellulose or PVDF) can be done by standard transfer protocols (17). In both cases, however, it is better to include SDS in the chosen buffer—we use Semidry Transfer Buffer (Recipe 17) and Wet Transfer Buffer (Recipe 18). PVDF membranes can be destained with Destaining Solution (Recipe 19), washed once with Western blot buffer, and then blocked with nonfat dry milk solution according to standard protocols. Western blotting can also be performed without the destaining step. Some antibodies do not work after a first-dimension BN-PAGE. In these cases, destaining does not help and a second-dimension SDS-PAGE has to be done.

Troubleshooting

Poor BN-PAGE Stacking Gel Wells

To separate very large MPCs—for example, the 26S proteasome—the separating gel contains very low percentages of acrylamide (around 3%). This makes removing the comb rather tricky, as it can disrupt the formation of the pockets. One option is to increase the acrylamide-bisacrylamide concentration of the stacking gel slightly (for example, by 0.3%). However, the difference between the acrylamide percentages of the stacking gel and the lower separating gel should be at least 0.5%. Another choice is to try to fix the wells with a syringe needle. Because these very low-percent acrylamide gels are more like "slime" than gel, just pushing the gel pieces into their proper position can sometimes "seal" the rupture. Unwanted air bubbles in the stacking gel can also be gently "sucked out" using a syringe. In these cases, before loading your sample, you can test for leakiness of the wells by applying the Cathode Buffer (Recipe 12) to one well and ensuring that it does not enter into the neighboring well.

Poor Sample Entry into Gel

One common problem is that Coomassie blue (and, with it, the Coomassie-bound proteins) precipitate in the gel pocket, and subsequently the sample does not enter the gel. The first critical step in BN-PAGE is the preparation of the sample, because cations that interact with Coomassie must be removed. Potassium and divalent cations must be substituted by 6-aminohexanoic acid to maintain the ionic strength of the solution necessary for the solubility and stabilization of many MPCs. Sodium ions are tolerated to a maximum concentration 20 to 50 mM. In addition, small unknown substances from cellular lysates can lead to precipitation of Coomassie blue (3), necessitating dialysis of the lysate. One simple solution if the Coomassie blue and bound protein precipitate is to load less sample onto the BN-PAGE gel. Try loading different volumes of the same sample--such as 20, 10, and 5 μl--to see which provides the best results. If even the smallest sample size causes precipitation, then dialysis of the sample should be improved. Use a larger dialysis reservoir volume, remove all air bubbles at the dialysis membrane, prolong dialysis time, or lower the ratio of sample volume to the area of dialysis membrane.

Peaks of Precipitation at the Gel Running Front

For certain detergents, especially Brij 96 and Triton X-100, the running front tends not to be a straight line, but peaks of precipitate may form. If the MPC of interest runs at higher positions than these precipitates, then this is not a problem. If the MPC comigrates with the precipitates at the running front, try reducing the amount of detergent in the BN-Dialysis Buffer (Recipe 4) or increasing the concentration of acrylamide. The latter leads to a better separation of the MPC of interest from the precipitated detergent.

Western Blotting Failure

Not all antibodies that are used for Western Blot experiments after SDS-PAGE will also work after a first-dimension BN-PAGE. Removal of the Coomassie blue from the Western blot membrane does not help, probably because the proteins are already on the membrane in a conformation that is not recognized by the antibody. In this case, the samples in the BN-PAGE gel must be run through a SDS-PAGE second-dimension gel to remove the Coomassie blue dye. It is best to verify Western blot results of first-dimension BN-PAGE by two-dimensional BN-PAGE and SDS-PAGE, to prove that the detected protein truly is the protein of interest and not a "background" band.

"Smearing" During BN-PAGE Separation

Instead of giving a defined band in the first dimension or a circular spot in two-dimensional BN-PAGE and SDS-PAGE, a smear generated in the first dimension may be seen. Smearing has several potential causes: The gel run might not have been perfect (that is, the voltage or temperature may have been too high, or the sample preparation not clean enough), the protein might have aggregated, or the protein might be present in several overlapping MPCs. Poor gel separation may be caused by the presence of cations or other small molecules that negatively influence the electrophoresis step. Improved dialysis of the sample should resolve this problem (see above). To avoid artifactual protein aggregation, try loading less sample and do not freeze-thaw the sample. Finally, if the protein is present in multiple MPCs, then BN-PAGE followed by another BN-PAGE (two-dimensional BN-PAGE) can determine whether several overlapping MPCs coexist that contain the protein of interest (5).

Lack of Reproducibility

Sometimes the protein of interest can be detected in a defined complex, but not in a reproducible manner. In this case, the MPC could be an artifact generated during electrophoresis. A gel with a distinct step in the gradient can cause an aggregation of protein at the step. When pouring the gradient, make sure that the flow of liquid is continuous and that both cylinders flow evenly. If the protein is not reproducibly detected following SDS-PAGE of the BN-PAGE sample, there may have been a small air bubble under the BN-PAGE gel slice when it was loaded onto the SDS-PAGE gel. The bubble prevents entry of protein along that position, producing a vertical line that gives the impression that two MPCs exist rather than one.

Only Monomers Detected

BN-PAGE can detect only MPCs of sufficient stability and abundance. The stability of the complex can be influenced by the detergent used to solubilize the proteins and load them onto the BN-PAGE gel. Thus, the choice of detergent is crucial for extracting but not disrupting the MPCs. Without detergent, proteins tend to aggregate during the stacking step of the electrophoresis and do not enter the separating gel properly. Unfortunately, general rules for the choice of detergents do not exist. In several cases, the abundance of several known MPCs was so low that they could not be detected by BN-PAGE (4). It is best to compare several detergents of different classes in preliminary experiments, including detergents that allow successful coimmunopurifications of the proteins expected to be present in a common MPC.

Lost Phosphorylation

During cell lysis or immunoprecipitations, orthovanadate is usually included to inhibit phosphatases. Because orthovanadate is a small molecule, it is rapidly separated from the sample during BN-PAGE. Furthermore, because it is a reversible inhibitor, phosphatases become active once orthovanadate is removed. Using pervanadate, an irreversible phosphatase inhibitor, in the BN-Dialysis Buffer (Recipe 4), should preserve protein phosphorylation during BN-PAGE.

Size Varies from Predicted

Many factors influence migration of proteins in polyacrylamide gels. In addition to these factors, it may be that the expected value is wrong and that the actual size is different from expected. Some of the factors that affect protein migration include the detergent micelle that forms around solubilized transmembrane MPCs and adds to MPC size. Therefore, the complex might be larger than that obtained by simply adding the molecular weights of the individual subunits. Other factors are protein glycosylation and phosphorylation, which also alter the electrophoretic behavior of proteins in BN-PAGE. In these cases, complexes usually appear larger than their actual molecular weights. Note that the marker proteins are nontransmembrane, nonglycosylated, and nonphosphorylated proteins.

Notes and Remarks

We present two published BN-PAGE experiments as examples. Details not mentioned here can be found in the relevant publications.

Analysis of the BCR by BN-PAGE Followed by Western Blotting

The purpose of this experiment was to investigate the influence of the detergent on the integrity of the oligomeric B cell antigen receptor (BCR) (6). The BCR consists of the antigen-binding membrane-bound immunoglobulin (mIg) and the signal-transducing disulfide-linked Ig-α/β heterodimer (Fig. 5A). The BCR was purified in the presence of various detergents from pervanadate-stimulated B cells by immunopurification with antibodies to phosphotyrosine residues and elution with phenylphosphate as described above. The samples were subjected to BN-PAGE, and the proteins were then visualized by Western blotting. In the presence of digitonin, one defined BCR complex was detected as shown by Western blotting with antibodies to mIg and antibodies to Ig-α/β (Fig. 5B, lanes 1 and 3). When the more stringent detergent SDS (1%) was added to the purified BCR, the complex disassembled into its two subunits, mIg and Ig-α/β (Fig. 5B, lanes 2 and 4). Samples were also prepared with different concentrations of the detergent thesit, and the purified BCRs were analyzed by BN-PAGE (Fig. 5C). In this experiment, a mutant BCR was used. At low concentrations of thesit, BCR dimers were detected that were not stable in higher concentrations of detergent. This experiment demonstrates the importance of the detergent in maintaining the integrity of MPCs. The detergent dependence of the stability of the T cell antigen receptor (TCR) is also well known (18). Our experience is that the choice of detergent is less critical for soluble MPCs than it is for transmembrane MPCs.

Fig. 5.

Example of one-dimensional BN-PAGE with subsequent Western blotting. (A) Schematic of the BCR. The BCR is an MPC consisting of a mIg and one covalently linked Ig-α/β heterodimer. (B) The BCR was purified from pervanadate-stimulated and digitonin-lysed B cells using antibodies to phosphotyrosine for immunopurification. The BCR was separated by BN-PAGE (5.5 to 16%), and Western blotting was performed using antibodies to mIg and Ig-α/β. The BCR was found as a single complex of around 500 to 600 kD (lanes 1 and 3). Treatment of the BCR with SDS led to its dissociation to the mIg and Ig-α/β subunits, which were separated from each other by BN-PAGE (lanes 2 and 4). (C) A mutant BCR was purified in the presence of varying concentrations of the detergent thesit. When 0.5% of the detergent thesit was used to solubilize this BCR, this receptor was found as a 500- to 600-kD MPC. Lowering the thesit concentrations led to the detection of a mutant BCR dimer. Our interpretation was that this mutant BCR existed as a dimer on the cell surface and that high thesit concentrations led to the disassembly of the BCR dimer into BCR monomers. Note that the wild-type BCR exists even in complexes larger than a dimer. [Reprinted from Immunity, Vol. 13, W. W. Schamel and M. Reth, Monomeric and oligomeric complexes of the B cell antigen receptor," pp. 5-14, copyright (2000), with permission from Elsevier]

Two-Dimensional BN-PAGE and SDS-PAGE with Silver Staining

The purpose of this experiment was the identification of abundant MPCs in total lysates of HEK293 cells (3). BN-PAGE samples were prepared and, as a control, an aliquot of the dialyzed lysate was treated with 1% SDS and heated to 95°C for 5 min before separation by BN-PAGE. This treatment ensures that all MPCs are disrupted before their separation, providing the positions of monomeric proteins in the two-dimensional gel. The proteins were visualized by silver staining. Monomeric proteins were localized to a hyperbolic-shaped diagonal (Fig. 6A). In contrast, proteins that were present in the same MPC were found as individual spots aligned in vertical columns (Fig. 6B; see also Fig. 1). Several proteins were subsequently identified by tandem mass spectrometry (3). Here, we indicate as examples the 20S proteasome, the small subunits of which are located on a vertical line (Fig. 6B; marked P), and the protein lactate dehydrogenase subunit B, which is present in three distinct complexes (Fig. 6B; marked B), two of which are more abundant and contain lactate dehydrogenase subunit A (Fig. 6B; marked A). This experiment demonstrates that endogenous MPCs can be identified by a two-dimensional BN-PAGE and SDS-PAGE approach.

Fig. 6.

Example of two-dimensional BN-PAGE and SDS-PAGE with subsequent silver staining. (A) The dialyzed lysate of HEK293 cells was prepared using the detergent Triton X-100 and subsequently treated with 1% SDS. MPCs were separated by two-dimensional BN-PAGE and SDS-PAGE (5.5 to 17% and 10%, respectively), and proteins were visualized by silver staining. Because MPCs were destroyed by SDS before the first dimension, only protein monomers are visible within the hyperbolic diagonal. (B) The dialyzed lysate of HEK293 cells was prepared using the detergent Triton X-100 without SDS treatment. MPCs are visible below and to the left of the diagonal. The 20S proteasome (P) and MPCs containing lactate dehydrogenase subunits A and B (A, B) are marked by arrows. [Reprinted from Molecular and Cellular Proteomics, Vol. 3, M. M. Camacho-Carvajal et al., "Two-dimensional blue native/SDS gel electrophoresis of multi-protein complexes from whole cellular lysates," pp. 176–182, copyright (2004), with permission from the American Society for Biochemistry and Molecular Biology]

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