Blue native electrophoresis (BN-PAGE, also known as blue native page) allows for the analysis of mitochondrial protein complexes in their native state. This protocol enables researchers to determine the size, abundance, and subunit composition of these complexes using Coomassie dye-based separation. In addition to resolving mature complexes, blue native electrophoresis can also detect assembly intermediates of mitochondrial complexes, providing insight into the stepwise assembly of these structures. The method involves a two-dimensional gel system, starting with native electrophoresis followed by SDS-PAGE for further resolution. BN-PAGE is ideal for studying multisubunit enzymes and protein interactions. Abcam’s detailed protocol includes sample preparation, gel casting, electrophoresis conditions, and immunodetection steps, making it a reliable resource for proteomics and mitochondrial research.

Introduction

BN-PAGE is a specialized electrophoresis technique that preserves protein complexes in their native form during separation. Unlike SDS-PAGE, which denatures proteins, BN-PAGE uses non-denaturing conditions and Coomassie dye to impart charge without disrupting structure. This allows for accurate analysis of intact mitochondrial complexes. Most proteins function as part of larger multiprotein complexes, which underscores the importance of techniques like BN-PAGE. The protocol is particularly useful in studying oxidative phosphorylation (OXPHOS) systems and other multisubunit assemblies. Saccharomyces cerevisiae is a widely used model organism for mitochondrial proteomics and complexome analysis. This protocol provides a step-by-step guide, from sample solubilization to blot development, ensuring reproducibility and clarity for researchers working in cell biology, biochemistry, and molecular diagnostics.

Background and principles

Blue native electrophoresis was first described by Schägger and von Jagow in 1991 as a method to separate protein complexes based on size and dye binding. Coomassie blue G binds to proteins, imparting a negative charge proportional to their mass. This enables separation in a polyacrylamide gel without denaturation. The protocol includes a first dimension native gel and an optional second dimension SDS-PAGE for subunit analysis. BN-PAGE is particularly suited for mitochondrial studies, where maintaining complex integrity is crucial. The technique combines biochemical precision with compatibility for downstream immunodetection, making it a cornerstone in proteomic workflows.

Reagents and equipment:

• Primary BN-PAGE tested antibody
• Secondary antibody which should be conjugated appropriately for the detection method of choice
• Electrophoresis and western blotting reagents
• 10% lauryl maltoside solution (n-dodecyl-β-D-maltopyranoside, ab109857)
• ​6-aminocaproic acid, Bis-Tris, Tricine
• Coomassie blue G
• Vertical acrylamide electrophoresis unit
• Electroblotting unit-fully submerged
• pH meter, weighing balance and other standard lab equipment

Buffer recipes:

Phosphate-buffered saline solution (PBS)​:

• ​1.4 mM KH₂PO₄
• 8 mM Na_​2HPO₄
• 140 mM NaCl
• 2.7 mM KCl, pH 7.3

Protease inhibitor stocks (each is 1000x)

• 1 M phenylmethanesulfonyl fluoride (PMSF) in acetone
• 1 mg/mL leupeptin
• ​1 mg/mL pepstatin

First dimension electrophoresis cathode buffer

• 50 mM Tricine
• ​15 mM Bis-Tris
• 0.02% Coomassie blue G
• Check pH and adjust to 7.0

First dimension electrophoresis anode buffer

• 50 mM Bis-Tris
• ​Check pH and adjust to 7.0

Second dimension electrophoresis running buffer

• 25 mM Tris
• ​192 mM glycine
• 0.1% SDS

SDS-PAGE denaturing buffer

• 10% glycerol
• 2% SDS
• 50 mM Tris, pH 6.8
• 0.002% Bromophenol blue
• 50 mM dithiothreitol

​Tris/glycine or Towbin electroblotting transfer buffer

• 25 mM Tris
• 192 mM glycine
• 10% methanol
• 0.1% SDS

Membrane washing buffer

• PBS plus 0.05% Tween 20

Membrane blocking buffer

• PBS plus 5% non-fat milk powder

Alkaline phosphatase color development buffer

• ​0.1 M diethanolamine (DEA)
• 5 mM MgCl​₂
• 100x NBT stock 50 mg/mL in 100% DMF
• 100x BCIP stock 50 mg/mL in 70% DMF
• ​DMF dimethylformamide

Buffer A

• 0.75 M 6-aminocaproic acid, 50 mM Bis-Tris/HCl, pH 7.0
• 1 µg/mL leupeptin
• ​1 µg/mL pepstatin
• 1 mM PMSF
• Stock leupeptin: 1 mg/mL (water)
• Stock pepstatin: 1 mg/mL (ethanol)
• Stock PMSF: 0.3 M (ethanol)
• LM: n-dodecyl-​​β-D-maltoside

Stage 1 - Sample preparation

Blue native polyacrylamide gel electrophoresis (BN-PAGE) is performed essentially as described by Schä​gger and von Jagow (1991), Analytical Biochemistry, 199, 223-31.

First, solubilized samples are stained with a charged (Coomassie) dye. The intact mitochondrial complexes are then separated by electrophoresis based upon how much dye was bound, which is proportional to their size. This first dimension gel can be immediately western blotted, or alternatively, the protein components of the resolved complexes can be further separated in a second dimension after soaking the gel in denaturing SDS buffer.

When performing blue native electrophoresis, it is always recommended to isolate mitochondria from cells before analysis. It is possible to probe whole tissue or cell extract but this may result in a weaker signal.

We also recommended optimizing sample concentration for your experiment.

Steps

Resuspend 0.4 mg of sedimented mitochondria in 40 µL 0.75 M aminocaproic acid, 50 mM Bis-Tris, pH 7.0.

Add 7.5 µL of 10% n-dodecyl-β-D-maltopyranoside.

Mix and incubate for 30 min on ice.

Centrifuge at 72,000 x g for 30 min.

• The Beckman Optima bench top ultracentrifuge is recommended for small sample volumes.
• However, a bench-top microcentrifuge at maximum speed, usually around 16,000 x g should suffice, although it is not ideal.

Collect supernatant and discard pellet.

Add 2.5 µL 5% solution/suspension of Coomassie blue G in 0.5 M aminocaproic acid to the supernatant.

Add protease inhibitors (e.g. 1 mM PMSF, 1 µg/mL leupeptin and 1 µg/mL pepstatin, see buffer recipes).

Stage 2 - Native acrylamide gel preparation and electrophoresis in the first dimension

Native acrylamide gels can be poured by hand. While it is possible to use a single acrylamide concentration such as a straight 10% gel, we highly recommend the use of a linear acrylamide concentration such as 6–13%. A recipe for pouring these native acrylamide gels in a 10-gel BioRad Mini-PROTEAN II multicasting chamber when using a two chamber gradient former is detailed below.

The acrylamide concentrations given in this procedure can be adjusted to optimize the separation of complexes of interest.

Steps

Pour native acryalmide gel by hand or by machine.

• Recipes for pouring these native acrylamide gels in a 10-gel BioRad Mini-PROTEAN II multicasting chamber, when using a two-chamber gradient former, are detailed below with recommended acrylamide - BioRad 30% Acrylamide/Bis Solution 37.5:1.

Once poured, cover the gels in 50% isopropanol solution.

When all 10 gels have set pour off the isopropanol, rinse with water and remove gels from casting chamber.

Use a stacking gel and comb.

Stacking gel recipe for 5 mL:

• 0.7 mL 30% acrylamide
• 1.6 mL dd water
• 0.25 mL 1 M Bis-Tris, pH 7.0
• 2.5 mL 1 M aminocaproic acid, pH 7.0
• 40 µL 10% APS
• ​10 µL TEMED

​Samples between 5–20 µL should be loaded into wells.

Electrophoresis conditions vary. However, the samples should be separated at 150 V for approximately 2 h or until the sample buffer blue dye has almost run off the bottom of the gel.

Stage 3 - Electrophoresis in the second dimension

The first dimension gel may be western blotted and the separated mitochondrial complexes probed with antibodies. If so, proceed to the next section.

Alternatively, the mitochondrial complexes can be further resolved into their protein subunit in a second (denaturing) dimension. Once complete, you can proceed with electroblotting as described below.

Steps

Cut each gel lane out of the first dimension gel and soak in SDS denaturing buffer.

• See our recipe for SDS denaturing buffer under recipes.

Each lane should be turned 90° and loaded onto the top of an SDS-PAGE 10-20% acrylamide gel.

Stage 4 - Electroblotting and immunodetection

Electroblotting should be performed with a fully submerged system such as BioRad Mini Trans-blot system. We recommend using the Tris-Glycine transfer method for blotting BN-PAGE gels (detailed in the buffers section).

We also highly recommend using a PVDF membrane such as Immobilon rather than nitrocellulose. Also, altering the electroblotting current and duration may improve the resolution and transfer of some proteins.

Steps

After electrophoresis the gel should be soaked in transfer buffer for 30 min before assembling the transfer sandwich.

Electroblotting should be carried out at 150 mA for 1.5 h.

Membranes should be blocked for at least 3 h in 5% milk/PBS solution.

Wash the membrane for 10 min in PBS 0.05% Tween 20.

Incubate the membrane with the primary BN-PAGE monoclonal antibody.

• Antibodies should be diluted to the recommended concentration in a 1% milk/PBS incubation solution.
• 5 mL of antibody solution should be enough to cover a 100 cm² membrane and constant rocking/agitation/rolling is recommended.

Wash the membrane in PBS 0.05% Tween 20 solution for 5 min.

• Repeat this step twice.

Incubate the membrane with the secondary antibody, which should be conjugated appropriately for the detection method of choice.

• Use this antibody at the dilution recommended by the manufacturer in a 1% milk/PBS solution.

Wash the membrane in PBS 0.05% Tween 20 solution for 5 min.

• Repeat this step twice.

Rinse the blot in PBS to remove any Tween 20 which may inhibit detection.

Stage 5 - Blot development

For more details, see the manufacturer's instructions.

Steps

The membrane should be incubated in AP color development buffer supplemented with 1% v/v BCIP and 1% v/v NBT.

Develop until a satisfactory signal is achieved.

Terminate development by rinsing the blot in water.

For more details, see the manufacturer's instructions.

Steps

The membrane should be incubated in HRP color development solution.

• Incubate for 2 min.

Cover the membrane with a transparent wrap/cling film and expose to X-ray film under appropriate darkroom conditions and film development.

Analysis of native complexes

The analysis of native complexes is fundamental for unraveling the intricate architecture and biological roles of protein complexes within cells. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a robust method for separating and characterizing native protein complexes, including challenging targets like membrane protein complexes and mitochondrial protein complexes. By maintaining the native structure of these assemblies, BN-PAGE enables researchers to assess not only the size and molecular weight of complexes but also their relative abundance and composition within biological samples.

A key advantage of BN-PAGE is its compatibility with downstream protein detection methods. After separation, protein bands corresponding to native complexes can be excised and subjected to mass spectrometry, allowing for the identification of individual proteins and post-translational modifications. This approach is particularly valuable for studying mitochondrial inner membrane protein complexes, such as the respiratory complexes involved in oxidative phosphorylation and cell survival. The use of acrylamide gradient gels enhances the resolution of complexes with varying molecular masses, while careful control of protein concentration and transfer buffer conditions ensures optimal protein separation and detection.

BN-PAGE is also instrumental in clinical diagnosis, as alterations in the composition or abundance of native complexes can serve as biomarkers for diseases, including neurodegenerative disorders. By analyzing mitochondrial protein complexes from patient samples, researchers can gain insights into disease mechanisms and identify potential diagnostic targets. The technique is equally valuable in basic research, where it supports the study of protein complex assembly, stability, and function in systems ranging from yeast strains and cell cultures to isolated mitochondria. When combined with affinity purification or gel filtration, BN-PAGE provides a comprehensive platform for dissecting the molecular details of native protein complexes, advancing our understanding of cellular processes and disease.

Comparison to other methods

Compared to SDS-PAGE, BN-PAGE offers the advantage of preserving protein complex integrity, allowing for functional and structural analysis. While SDS-PAGE separates proteins by molecular weight alone, BN-PAGE maintains native interactions, making it ideal for studying multisubunit assemblies. Isoelectric focusing (IEF) provides high resolution but requires a more complex setup and is less suited for membrane proteins. BN-PAGE also surpasses clear native PAGE (CN-PAGE) in sensitivity due to Coomassie dye’s charge-enhancing properties.

Applications and downstream protein detection methods

BN-PAGE is widely used in mitochondrial research, particularly for analyzing OXPHOS complexes. It enables the study of protein-protein interactions, complex stoichiometry, and post-translational modifications. The technique is applicable in disease research, such as neurodegeneration and metabolic disorders, where mitochondrial dysfunction is implicated. It also supports proteomic profiling and biomarker discovery. This protocol facilitates integration with western blotting, allowing for specific antibody-based detection. Researchers can apply BN-PAGE to cell lines, tissues, and purified organelles, making it a flexible tool for both basic and translational science.

Limitations

Despite its strengths, BN-PAGE has limitations. The technique requires careful sample preparation to avoid complex dissociation. Coomassie dye may interfere with downstream mass spectrometry unless removed. Gel casting and gradient formation can be technically demanding, and resolution may vary depending on acrylamide concentration. Additionally, whole-cell extracts may yield weaker signals compared to isolated mitochondria. Optimization of antibody concentrations and electrophoresis conditions is often necessary. Abcam’s protocol addresses these challenges with detailed buffer recipes and troubleshooting tips, but users should be prepared for iterative refinement to achieve optimal results.

Troubleshooting

Common issues in BN-PAGE include weak western blot signals, blue background on gels, and poor resolution of complexes. To improve signal strength, ensure mitochondrial isolation, and optimize sample concentration. Use fresh protease inhibitors and verify buffer pH. Blue background may result from excess Coomassie dye—reduce dye concentration or extend washing steps. If protein transfer is inefficient, adjust the electroblotting current and duration. Abcam recommends using PVDF membranes and optimizing antibody dilutions. For gradient gels, ensure proper acrylamide mixing and polymerization. Their troubleshooting guide provides practical solutions to maintain consistency and clarity in results.