Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE)
BN-PAGE is suitable for separation of water soluble as well as membrane-bound protein complexes in the range 10 to 1000 kDa (resolution in the range 10-100 kDa is significantly lower). The separated protein complexes are in their native conformation, and therefore sustain their enzymatic activity and oligomeric state. Thus this method is also used for determination of subunit composition and degree of homogeneity of protein complexes.
In this method a charge shift on the proteins is induced by negatively charged Coomassie dye instead of SDS! And 6-Aminocaproic acid improves the solubilization of proteins.
Cathode buffer, pH 7.0 at 4 C (no need to adjust!):
50 mM Tricine (Sigma, T5816, 179.2 g/mol) 8.96 g
15 mM BisTris (Sigma, B9754, 209.2 g/mol) 3.138 g
0.02 Coomassie blue G-250 200 mg
add H2O to 1000 ml.
Store at low temperature but bear in mind that Coomassie blue may aggregate.
Anode buffer, pH 7.0 at 4 C with HCl:
50 mM BisTris (Sigma, B9754, 209.2 g/mol) 10.46 g
add H2O to 1000 ml.
Store at low temperature.
3 Gel buffer, pH 7.0 at 4 C with HCl:
150 mM BisTris (Sigma, B9754, 209.2 g/mol) 3.138 g
1.5 M 6-Aminocaproic acid (Sigma, A2504, 131.2 g/mol) 19.68 g
add H2O to 100 ml.
Store at low temperature.
Sample buffer, pH 7.0 at 4 C with HCl:
This buffer is used for water-soluble proteins!
75 mM BisTris (Sigma, B9754, 209.2 g/mol) 1.569 g
750 M 6-Aminocaproic acid (Sigma, A2504, 131.2 g/mol) 9.84 g
10 Glycerol (87) 11.49 ml
add H2O to 100 ml.
Store at low temperature.
There are some modifications when we have membrane-bound proteins:
Sample buffer A: Sample buffer plus 1 Triton X-100 (0.1 g/10 ml);
Sample buffer B: Sample buffer plus 5 Coomassie bue G-250 (0.5 g/10 ml).
In this case solubilize the protein sample in Sample buffer A (e.g. 20 l), and add some of Sample B (e.g. 1 l), thus reaching a detergent/Coomassie ratio of 4:1 (g/g).
Alternatively it is also possible to use imidazole instead of BisTris!
Cathode buffer, pH 7.0 at 4 C (no need to adjust!):
50 mM Tricine (Sigma, T5816, 179.2 g/mol) 8.96 g
7.5 mM Imidazole (Sigma, I0125, 68.08 g/mol) 0.51 g
0.02 Coomassie blue G-250 200 mg
add H2O to 1000 ml.
Store at low temperature but bear in mind that Coomassie blue may aggregate.
Anode buffer, pH 7.0 at 4 C with HCl:
25 mM Imidazole (Sigma, I0125, 68.08 g/mol) 1.7 g
add H2O to 1000 ml.
Store at low temperature.
3 Gel buffer, pH 7.0 at 4 C with HCl:
75 mM Imidazole (Sigma, I0125, 68.08 g/mol) 0.51 g
1.5 M 6-Aminocaproic acid (Sigma, A2504, 131.2 g/mol) 19.68 g
add H2O to 100 ml.
Store at low temperature.
Sample buffer, pH 7.0 at 4 C with HCl:
This buffer is used for water-soluble proteins!
37.5 mM Imidazole (Sigma, I0125, 68.08 g/mol) 0.255 g
750 M 6-Aminocaproic acid (Sigma, A2504, 131.2 g/mol) 9.84 g
10 Glycerol (87) 11.49 ml
add H2O to 100 ml.
Store at low temperature.
Resolving gel (10 ml for two 7.5 10 cm gels of 0.75-1 mm thickness):
|
|
5 |
6.5 |
Final concentration |
||
3 Gel buffer |
3.33 ml |
3.33 ml |
50 mM BisTris, and 500 mM 6–Aminocaproic acid |
||
Acrylamide, 30 |
1.7 ml |
2.2 ml |
|
|
|
Glycerol, 87 |
0.6 ml |
0.6 ml |
5 |
|
|
|
H2O |
4.26 ml |
3.76 ml |
|
|
|
TEMED |
10 l |
10 l |
|
|
|
|
10 APS |
100 l |
100 l |
|
|
|
Stacking 3.3 gel (5 ml for 2 gels):
3 Gel buffer 1.665 ml
Acrylamide, 30 0.55 ml
H2O 2.73 ml
TEMED 5 l
10 APS 50 l
Note that you may also use a gradient gel to separate the protein complexes: for complexes of 300 kDa and greater the 5-13 gradient should work fine.
Molecular weight markers (HMW Calibration Kit, Pharmacia and other sellers):
|
Protein marker |
Mol. Wt. (kDa) |
Subunit Mol. Wt. (kDa) |
|
Thyroglobulin |
669 |
2 330 |
|
Ferritin (Sigma, A4503) |
880, 2 440 |
2 220 (also 18.5) |
|
Apoferritin (Sigma, A3641) |
481.2 |
24 20 |
|
Catalase |
232 |
2 60 |
|
Lactate dehydrogenase |
140 |
4 35 |
|
Albumin |
132 |
2 66 |
|
Aldehyde dehydrogenase |
216-240 |
4 54-60 |
|
Glutamate dehydrogenase |
310-350 |
6 55.4 |
|
-Amylase |
200 |
|
|
Aldolase |
158 |
|
|
Hexokinase |
100 |
|
|
Creatine kinase |
86 |
2 43 |
|
Hemoglobin |
68 |
4 17 |
|
Pyruvate kinase |
58 |
|
Perform the BN-PAGE in a cold room. Start at 100 V and keep it until the proteins enter the resolving gel. Thereafter the voltage has to be increased to 250 V. Current, during the electrophoresis process, should not exceed 15 mA! Run the gel until the blue front reaches the bottom of the gel.
When the run is completed cut out the lanes and soak them in the SDS sample buffer (with 5-mercaptoethanol) for 30 min at room temperature. The sample is ready for the SDS-PAGE (second dimension).
It is also possible to store the BN-PAGE lanes at -70C before soaking in the SDS sample buffer.
Note that if the gel is intended for the immunoblotting in such a case change the blue cathode buffer to the cathode buffer made without Coomassie blue after 1/2-2/3 top of the gel becomes coloured in blue. The chamber should not be washed! Changing the buffer decreases the amount of Coomassie blue in transfer and helps for Western blot analysis.
Literature:
-
Schagger H, von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem., 1991, 199: 223-231.
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Schagger H, Cramer W, von Jagow G. Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem., 1994, 217: 220-230.
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Schagger H. Native gel electrophoresis. In “A practical guide to membrane protein purification”, Academic Press 1994, 81-104.
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Brookes PS, Pinner A, Ramachandran A, Coward L, Barnes S, Kim H, Darley-Usmar VM. High throughput two-dimensional blue-native electrophoresis: A tool for functional proteomics of mitochondria and signaling complexes. Proteomics, 2002, 2: 969-977.
2008 02 25 – Julius