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This protocol for blue native electrophoresis is designed for use with the following products:
Phosphate buffered saline solution (PBS)
1.4 mM KH2PO4
8 mM Na2HPO4
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
SDS-PAGE denaturing buffer
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
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 MgCl2
100x NBT stock 50 mg/mL in 100% DMF
100x BCIP stock 50 mg/mL in 70% DMF
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)
Blue native polyacrylamide gel electrophoresis (BN-PAGE) is performed essentially as described by Schägger 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. We offer monoclonal antibodies for the detection of all five OXPHOS complexes simultaneously (ab110412) or each of the OXPHOS complexes individually.
When performing blue native electrophoresis, it is always recommended to isolate mitochondria from cells before analysis. The following kits can be used:
It is possible to probe whole tissue or cell extract but this may result in a weaker signal.
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.
|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|
The first dimension gel may be western blotted and the separated mitochondrial complexes probed with antibodies. If so, proceed to the next section. As an alternative the mitochondrial complexes can be further resolved into their protein subunit in a second (denaturing) dimension. To do this:
Electroblotting should be performed with a fully submerged system such as BioRad Mini Trans-blot system. We recommends using the Tris-Glycine transfer method for blotting BN-PAGE gels. The recipes for all buffers are detailed in the buffers section. Also highly recommended is the use of a PVDF membrane such as Immobilon rather than nitrocellulose membrane.
Blot development with an alkaline phosphatase conjugated secondary antibody
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.
Blot development with a horseradish peroxidase conjugated secondary antibody
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. 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. For more details see the manufacturer's instructions.
It is always recommended to optimize sample concentration.
Gel acrylamide concentrations and transfer
The acrylamide concentrations given in this procedure can be adjusted to optimize separation of complexes of interest. Also altering electroblotting current and duration may improve resolution and transfer of some proteins.
The primary antibody should be used at the recommended concentration provided on the online datasheet. However, when using low sample loads or particularly when analyzing alternative species as a source of material, some optimization may be necessary (usually involving increasing the concentration of the primary antibody). 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.
After electrophoresis, the gel or blot has a blue background
Once the first dimension separation is almost complete, the cathode dye containing Coomassie blue G can be replaced by cathode buffer without dye. Further electrophoresis will remove most of the dye from the gel.
Weak or no western blotting signal
To check the transfer, stain the blot after transfer with Ponceau Red. Pre-stained markers confirm good transfer. Over transfer or "blow through" may occur. Reduce transfer current or time, or use a membrane with smaller pore size or put a second membrane behind first as precaution.
For quick reference only. We recommend becoming familiar with previous details of this protocol document before performing the assay.