BN-PAGE Protocol
Licia Miller Product Manager
Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an electrophoretic technique used to separate and analyze large protein complexes. It preserves protein complexes in their native state under non-denaturing conditions by treating samples with mild detergents and negatively charged dyes such as Coomassie Blue G-250.
During electrophoresis, these complexes are separated according to their size and the degree of binding to the dye, allowing the distribution of different protein complexes to be clearly observed in the gel. BN-PAGE is particularly suitable for studying membrane proteins and protein assemblies that need to remain active because it can separate them without destroying their structure.
BN-PAGE technology is favored because it can provide high-resolution protein complex separation. It can be used not only for protein complex analysis in basic research, but also for quality control of protein drugs in the biopharmaceutical field. Through BN-PAGE, researchers can better understand the interactions between proteins and their functions in cells.
Required reagents and equipment:
- BN-PAGE validated primary antibodies
- Suitable conjugated secondary antibodies
- Electrophoresis and Western Blotting Reagents
- 10% Lauryl Maltoside Solution (n-dodecyl-β-D-maltopyranoside)
- 6-Aminocaproic acid, Bis-Tris, Tricine
- Coomassie Brilliant Blue G (example B104241)
- Vertical acrylamide electrophoresis apparatus
- Electroblotting Apparatus - Fully Immersed
- pH meter, balance and other standard laboratory equipment
Buffer formulation:
Phosphate Buffered Saline (PBS) :
- 1.4 mM KH2PO4
- 8 mM Na2HPO4
- 140 mM NaCl
- 2.7 mM KCl, pH 7.3
Protease inhibitor stock solution (1000× each) :
- 1 M phenylmethanesulfonyl fluoride (PMSF) in acetone
- 1 mg/mL Pepstatin
First dimension electrophoresis cathode buffer:
- 50 mM Tricine
- 15 mM Bis-Tris
- 0.02% Coomassie Brilliant Blue G
Adjust pH to 7.0
First dimension electrophoresis anode buffer:
- 50 mM Bis-Tris
Adjust pH to 7.0
Second dimension electrophoresis buffer:
- 25 mM Tris
- 192 mM Glycine
- 0.1% SDS
SDS-PAGE Denaturation Buffer:
- 10% glycerin
- 2% SDS
- 50 mM Tris, pH 6.8
- 0.002% Bromophenol Blue
- 50 mM dithiothreitol (DTT)
Tris/Glycine or Towbin Electroblotting Buffer:
- 25 mM Tris
- 192 mM Glycine
- 10% Methanol
- 0.1%SDS
Membrane Wash Buffer:
- PBS with 0.05% Tween 20
Membrane Blocking Buffer:
- PBS plus 5% skim milk powder
Alkaline phosphatase color development buffer:
- 0.1 M diethanolamine (DEA)
- 5 mM MgCl2
- 100× NBT stock solution, dissolved in 100% DMF, concentration 50 mg/mL
- 100× BCIP stock solution, dissolved in 70% DMF, concentration 50 mg/mL
Buffer A:
- 0.75 M 6-aminohexanoic acid, 50 mM Bis-Tris/HCl, pH 7.0
- 1 µg/mL Leupeptin
- 1 µg/mL Pepstatin
- 1 mM PMSF
- Leupeptin stock solution: 1 mg /mL (water)
- Pepstatin stock solution: 1 mg /mL (ethanol)
- PMSF stock solution: 0.3 M (ethanol)
- LM: n-Dodecyl-β-D-maltoside
Phase 1 Sample preparation
The BN-PAGE experiments were performed basically according to the method described by Schägger and von Jagow (1991) .
First, the solubilized sample is stained with a charged (Coomassie Brilliant Blue) dye. The intact mitochondrial complexes are then separated by electrophoresis according to the amount of dye bound, which is proportional to their size. This first- dimension gel can be immediately subjected to Western blotting, or, after soaking the gel in a denaturing SDS buffer, the protein components of the separated complexes can be further separated in a second dimension.
When whole tissue or cell extracts are used as samples, the signal is often weak, so it is recommended to separate mitochondria from cells in advance when performing BN-PAGE.
Experimental Steps
1. Resuspend 0.4 mg of mitochondrial pellet in 40 µL of 50 mM Bis-Tris, pH 7.0, containing 0.75 M aminocaproic acid.
2. Add 7.5 µL of 10% n-dodecyl-β-D-maltopyranoside. Mix well and incubate on ice for 30 minutes.
3. Centrifuge at 72,000 xg for 30 minutes.
Tip: We recommend using a benchtop ultracentrifuge, but if you do not have one, you can also centrifuge at the maximum speed of a benchtop microcentrifuge (usually around 16,000 xg) .
4. Collect the supernatant and discard the precipitate.
5. Add 2.5 µL of 5% Coomassie Brilliant Blue G solution/suspension (dissolved in 0.5 M aminocaproic acid) to the supernatant.
6. Add protease inhibitors (e.g. 1 mM PMSF, 1 µg/mL Leupeptin, and 1 µg/mL Pepstatin, see Buffer Recipes) .
Phase 2 Preparation of native acrylamide gel and first-dimension electrophoresis
Native acrylamide gels can be poured manually. Although it is possible to use a single acrylamide concentration, such as a 10% gel, it is generally more advisable to use linear acrylamide concentrations, such as 6-13%.
Experimental Steps
1. Pour natural acrylamide gel by hand or machine.
When using a dual chamber gradient former, the recipe for pouring these native acrylamide gels into a 10-gel BioRad Mini-PROTEAN II multiplex perfusion chamber is detailed below. The recommended acrylamide is BioRad 30% acrylamide/ Bis solution (37.5:1) .
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2. After pouring, cover the gel with 50% isopropyl alcohol solution.
3. When all 10 gels have solidified, pour off the isopropyl alcohol, rinse with water and remove the gels.
4. Use a thickening gel and a comb.
5 mL stacking gel recipe:
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5. Load 5-20 µL of sample into the wells. Set appropriate electrophoresis conditions. Samples should be V for approximately 2 hours or until the sample buffer blue dye has almost washed out from the bottom of the gel.
Stage 3 Second Dimension Electrophoresis
The gel obtained by the first dimension electrophoresis can be used for protein blot analysis. If antibodies are needed to detect the isolated mitochondrial complexes, this step can be skipped and the next step can be entered.
If it is desired that the mitochondrial complexes can be further resolved into their protein subunits in the second dimension (denaturation), this procedure can be followed and electroblotting performed.
Experimental Steps
1. Cut each gel lane from the first gel and soak in SDS denaturing buffer.
2. Rotate each lane 90° and load onto the top of an SDS-PAGE 10-20% acrylamide gel.
Tip: The gel should be wider to accommodate the first dimension gel strip.
Phase 4 Electroblotting and immunodetection
Electroblotting should be performed using a fully immersion system, such as the BioRad Mini Trans-blot system. We recommend using the Tris-Glycine transfer method for BN-PAGE gels (see the buffer section for details). In addition, it is recommended to use PVDF membranes (such as Immobilon) instead of nitrocellulose. In addition, changing the electroblotting current and duration may improve the resolution and transfer of some proteins.
Experimental Steps
1. After electrophoresis, the gel should be soaked in transfer buffer for 30 minutes and then the transfer sandwich should be assembled.
2. Electroblotting should be performed at 150 mAmp for 1.5 hours. Complete transfer of blue dye from the gel to the membrane indicates good electrophoretic transfer.
3. The membrane should be blocked in 5% milk/PBS solution for at least 3 hours, or overnight at 4°C.
4. Wash the membrane with PBS 0.05% Tween 20 for 10 minutes.
5. Dilute the primary antibody with 1% milk/PBS solution according to the recommended ratio, and incubate the membrane with BN-PAGE monoclonal antibody. Generally, 5 mL of antibody solution is enough to cover 100 cm2 of membrane. It is recommended to place it on a shaker and shake it continuously.
Note: Primary antibodies should be used at the recommended concentrations provided on the datasheet. However, some optimization (usually increasing the concentration of the primary antibody) may be necessary when working with low sample amounts or especially when analyzing alternative species as a source of material.
6. Wash the membrane twice with PBS 0.05% Tween 20 solution, 5 minutes each time.
7. Dilute the secondary antibody to the recommended concentration in PBS containing 1% milk/ BSA and incubate the membrane with the secondary antibody. The secondary antibody should be conjugated according to the selected detection method.
Note: The addition of 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. Generally, a dilution of 1:1000 - 10,000 is normal for enzyme-linked secondary antibodies.
Two methods that are highly recommended are alkaline phosphatase (AP) and horseradish peroxidase-conjugated secondary antibodies.
8. Wash the membrane twice with PBS 0.05% Tween 20 solution, 5 minutes each time.
9. Rinse the blot with PBS to remove Tween 20 that may inhibit detection.
Phase 5 Blot development
● Alkaline phosphatase conjugated secondary antibody
Experimental Steps
1. Incubate the membrane in AP development buffer containing 1% v/v BCIP and 1% v/v NBT.
2. Develop until a satisfactory signal is obtained.
3. Rinse the blot with water to stop development.
● Horseradish peroxidase-conjugated secondary antibody
Experimental Steps
1. Place the membrane in HRP colorimetric solution (ECL system is usually recommended , where the solution is 40:1 reagent A:B) and incubate for 2 minutes.
2. Cover the membrane with transparent plastic wrap and expose to X-ray film under appropriate darkroom conditions and develop the film.
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