Blue native electrophoresis

Last edited Fri 26 Jun 2015

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 mAmp 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 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.