Blue native electrophoresis

Determine the size, relative abundance, and subunit composition of mitochondrial protein complexes.

Last edited Fri 26 Jun 2015

Reagents and equipment:

Buffer recipes:

Phosphate-buffered saline solution (PBS)​:

Protease inhibitor stocks (each is 1000x)

First dimension electrophoresis cathode buffer

First dimension electrophoresis anode buffer

Second dimension electrophoresis running buffer

SDS-PAGE denaturing buffer

​Tris/glycine or Towbin electroblotting transfer buffer

Membrane washing buffer

Membrane blocking buffer

Alkaline phosphatase color development buffer

Buffer A

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.

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.

For 38 mL
For 32 mL
6% acrylamide
13% acrylamide
7.6 mL 30% acrylamide
14 mL 30% acrylamide
9 mL dd water
0.2 mL dd water
19 mL 1 M aminocaproic acid, pH 7.0
16 mL 1 M aminocaproic acid, pH 7.0
1.9 mL 1 M Bis-Tris, pH 7.0
1.6 mL 1 M Bis-Tris, pH 7.0
200 µL 10% APS
200 µL 10% APS
20 µL TEMED
20 µL TEMED
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%.

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:

​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.

A recipe for BN-PAGE anode and cathode electrophoresis running buffers are described in the buffer recipes section.

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.

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

This gel should be wider to accommodate the first dimension gel strip.

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.

A good electrophoretic transfer is indicated by the complete transfer of blue dye from the gel onto the membrane.

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

Blocking overnight at 4°C is recommended.

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

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

The primary antibody should be used at the recommended concentration provided on the datasheet. However, when using low sample loads or particularly when analyzing alternative species as a source of material, some optimization may be necessary (usually increasing the concentration of the primary antibody).

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

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

Including sodium azide as a preservative in this solution, or subsequent solutions, will inhibit the activity of horseradish peroxidase conjugated antibodies.

Secondary antibodies also vary and should be optimized for your system. Typically, a 1:1000–10,000x dilution is normal for commercially available enzyme-conjugated secondary antibodies.

Two highly recommended methods are alkaline phosphatase (AP) and horseradish peroxidase conjugated secondary antibodies.

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

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.

We highly recommend the ECL system where the solution is 40:1 reagent A:B.

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