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Gabrielle M. Siegers, Susana Minguet, Bernd Wollscheid and Wolfgang W. A. Schamel (25 July 2006) Sci. STKE 2006 (345), pl4. [DOI: 10.1126/stke.3452006pl4]
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PROTOCOL
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for the Identification and Analysis of Multiprotein Complexes
Mahima Swamy,1 Gabrielle M. Siegers,1 Susana Minguet,1 Bernd Wollscheid,2 and Wolfgang W. A. Schamel1*
(Published 25 July 2006)
TROUBLESHOOTING
Poor BN-PAGE Stacking Gel Wells Poor Sample Entry into Gel Peaks of Precipitation at the Gel Running Front Western Blotting Failure Smearing During BN-PAGE Separation Lack of Reproducibility Only Monomers Detected Lost Phosphorylation Size Varies from Predicted
1Max
Planck-Institut fr Immunbiologie und Universitt Freiburg, Biologie III, Stbeweg 51, D-79108 Freiburg, Germany 2NCCR Neuro Center for Proteomics, Institute for Molecular Systems Biology ETH Hnggerberg, Wolfgang Pauli-Str. 16, CH-8093 Zrich, Switzerland. *Corresponding author. Telephone, 49-761-5108-313; fax, 49-761-5108-423; e-mail, schamel@immunbio.mpg.de
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ABSTRACT
Multiprotein complexes (MPCs) play crucial roles in cell signaling. Two kinds of MPCs can be distinguished: (i) Constitutive, abundant MPCsfor example, multisubunit receptors or transcription factors; and (ii) signalinduced, transient, low copy number MPCsfor 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
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-!B (NF-!B), which is inhibited by binding to its inhibitor I!B; 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. Schgger for general applicability (3) and used it to study signaling proteins (36).
BN-PAGE
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 twodimensional 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. 176182, 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 SLP76 (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
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BN-PAGE
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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 ("-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 #-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.
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Marker Proteins
Aldolase (Sigma-Aldrich) Bovine serum albumin (BSA) (Sigma-Aldrich) Catalase (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).
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10%
Detergent
Digitonin or Triton X-100 or Brij 96 or Dodecylmaltoside 0.1 to 0.5% 0.1 to 0.5% 0.1 to 0.5% 0.5 to 1.0%
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 20C). Protease and phophatase inhibitors should be added immediately before use. Upon addition of orthovanadate, the buffer will become yellowish in color.
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Detergent
Digitonin or Triton X-100 or Brij 96 or Dodecylmaltoside 0.1% 0.1% 0.1% 0.3 to 0.5%
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.
Detergent
Digitonin or Triton X-100 or Brij 96 or Dodecylmaltoside 0.1 to 0.5% 0.1 to 0.5% 0.1 to 0.5% 0.5 to 1.0%
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 20C). Protease and phophatase inhibitors should be added immediately before use.
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Adjust pH to 7.0 with HCl. Store at 4C. 15 ml of BN-Gel Buffer are sufficient for one 30-ml gel.
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.
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.
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.
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Prepare 1 liter as a 10 stock, adjust pH to 7.0 with HCl, and store at 4C. Dilute 1:10 with dH2O before use.
Mix in the order listed and then incubate 5 to 30 min at room temperature until solution becomes brownish.
Adjust pH to 7.0 with HCl. Store at 4C. Note: Molecular weight markers are also commercially available from several sources, including Invitrogen or Pharmacia.
Reagent
Tris SDS Glycerol Bromophenol blue
Concentration
12.5 mM 4% 20% 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 #-mercaptoethanol. SDS as a powder and #-mercaptoethanol are toxic; therefore, use gloves and work under a hood.
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Reagent
Tris Glycine Methanol SDS
Concentration
48 mM 39 mM 20% 0.1%
Reagent
Tris Glycine Methanol SDS
Concentration
25 mM 190 mM 20%
0.1%
Instructions
PROTOCOL
4. Incubate on ice for 15 min. 5. Centrifuge at 13,000g for 15 min at 4C. 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. 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 4C. 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-&l 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).
C D
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.
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 4C 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 4C for at least 6 hours with continuous stirring.
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 2 107 cells and pellet by centrifugation at 350g for 5 min at 4C. 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 4C. 3. Resuspend the cell pellet at 2 107 cells per 1 &l of ice-cold Lysis Buffer (Recipe 5). 4. Incubate for 15 min on ice. 5. Centrifuge at 13,000g for 15 min at 4C. 6. Transfer the supernatant from step 5 into a new chilled tube. 7. Add 10 &l 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 4C. 9. Wash the beads three times with 1 ml of ice-cold BN-Dialysis Buffer (Recipe 4), centrifuging 400g for 2 min at 4C.
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10. Add 40 &l of ice-cold BN-Dialysis Buffer Containing Phenylphosphate (Recipe 6) and resuspend the beads using a 20-&l 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 4C 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 4 107 cells per 100 &l of BN-Lysis Buffer. 4. Incubate for 1 hour at 4C, resuspending the pellet every 15 min. 5. Centrifuge at 20,000g for 10 min at 4C. 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.
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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.
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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.
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.
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).
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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 (mg 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 bufferwe 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.
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Troubleshooting
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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.
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.
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.
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.06
.03
880
Ig-/
440 230
.5
mIg
.015
- +
- + : SDS
: % thesit
Ig-/ : WB
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-'/# 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-'/#. 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-'/# 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. 514, copyright (2000), with permission from Elsevier]
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.
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A
kD 175 83 top 880
B
kD 175 83 63
SDS-PAGE
+ SDS
63 47 32 25
SDS-PAGE
47 32 25
B A
P
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 twodimensional 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. 176182, copyright (2004), with permission from the American Society for Biochemistry and Molecular Biology]
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.
References and Notes
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15. G. Rigaut, A. Shevchenko, B. Rutz, M. Wilm, M. Mann, B. Seraphin, A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17, 10301032 (1999). 16. Properties of Detergents (Amphiphiles), as seen July 2006 (http://psyche.uthct.edu/shaun/SBlack/detergnt.html) and Table of Detergents, as seen July 2006 (http://www.abrf.org/ABRFNews/1997/December1997/dec97Table.html) from the Association of Biomolecular Resource Facilities. 17. Cell Biology, A Laboratory Handbook. Vols. 2 and 4. J. E. Celis, Ed. (Academic Press, San Diego, CA, 1998). 18. B. Alarcon, M. Swamy, H. M. van Santen, W. W. Schamel, T cell antigen receptor stoichiometry: Pre-clustering for sensitivity. EMBO Rep. 7, 490495 (2006). 19. We thank M. Reth and B. Alarcn for their scientific support. We also acknowledge financial support by the Deutsche Forschungsgemeinschaft through the Emmy Noether program (SCHA 976/1) and the SFB620 and the European Union founded grant EPI-PEP-VAC.
Citation: M. Swamy, G. M. Siegers, S. Minguet, B. Wollschied, W. W. A. Schamel, Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and analysis of multiprotein complexes. Sci. STKE 2006, pl4 (2006).
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