What Are the Procedures for Blue Native PAGE and Two-Dimensional BN-SDS-PAGE, and How Are Anodic and Cathodic Buffers Used?
Blue Native PAGE (BN-PAGE)
1. Experimental Materials and Buffers
(1) Sample buffer (cathodic buffer): 50 mM Bis-Tris (pH 7.0), 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S, and an appropriate amount of Coomassie Blue G-250 to confer a uniform negative charge on protein complexes.
(2) Loading buffer (anodic buffer): 5% Coomassie Brilliant Blue G-250, 500 mM 6-aminohexanoic acid, 100 mM Bis-Tris (pH 7.0).
(3) Gel solutions and running buffer for BN-PAGE: Typically composed of 15 mM Bis-Tris (pH 7.0) and 50 mM Tricine, used for both separation and stacking gels, as well as for electrophoresis.
2. Procedure
(1) Sample preparation: Protein complexes are mixed with a buffer containing Coomassie G-250, which helps maintain their native, non-denatured state and imparts a consistent negative charge for electrophoretic migration.
(2) Gel preparation: Prepare stacking and separation gels at concentrations suitable for the molecular weight and complexity of the target protein.
(3) Sample loading and electrophoresis: Load the prepared samples onto the gel and run electrophoresis at 4°C using the designated BN-PAGE running buffer.
(4) Staining and detection: After electrophoresis, visualize the protein bands by staining with Coomassie Brilliant Blue or other suitable protein-specific stains.
Two-Dimensional BN-SDS-PAGE
Two-dimensional BN-SDS-PAGE builds upon Blue Native PAGE by introducing a second dimension of separation using SDS-PAGE. This allows for the resolution of individual components within protein complexes initially separated under native conditions.
1. Second-Dimension Materials and Buffers
(1) SDS-PAGE sample buffer: Contains SDS and dithiothreitol (DTT) and is used to denature protein complexes prior to their separation in the second dimension.
2. Procedure
(1) First-dimension BN-PAGE: Conducted as previously described, under non-denaturing conditions using BN-PAGE to separate intact protein complexes.
(2) Preparation for second dimension: Excise the gel strip resulting from the first-dimension BN-PAGE and incubate it in SDS-containing sample buffer to facilitate the penetration and binding of SDS to the proteins.
(3) Second-dimension SDS-PAGE: Place the SDS-treated gel strip onto an SDS-PAGE gel and perform electrophoresis. This step resolves the subunits of the protein complexes according to their molecular weight.
(4) Staining and detection: Similar to BN-PAGE, protein bands are visualized using Coomassie Brilliant Blue or silver staining methods.
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