Want to understand the blue native page experiment and the steps of two-dimensional BN-SDS-PAGE, as well as the positive and negative buffers.
1. Blue Native PAGE (BN-PAGE)
Experimental Materials and Solutions
- Sample Buffer (Negative Buffer): 50 mM Bis-Tris (pH 7.0), 50 mM NaCl, 10% (w/v) glycerol, 0.001% Ponceau S, with an appropriate amount of Coomassie Blue G-250 added to maintain the negative charge of proteins.
- Positive Buffer (Loading Buffer): 5% Coomassie Brilliant Blue G-250, 500 mM 6-aminohexanoic acid, 100 mM Bis-Tris (pH 7.0).
- Separation Gel and Aggregation Gel Solution, and BN-PAGE Running Buffer: Typically contains 15 mM Bis-Tris (pH 7.0) and 50 mM Tricine.
Steps:
1. Sample Preparation: Treat samples in a buffer containing Coomassie G-250 to maintain the non-denatured state of the protein complex.
2. Gel Preparation: Prepare separation gel and aggregation gel at appropriate concentrations depending on the size and complexity of the target protein.
3. Sample Loading and Electrophoresis: Load the samples onto the gel and perform electrophoresis at 4°C using BN-PAGE running buffer.
4. Staining/Detection: After electrophoresis, stain and detect the proteins using Coomassie Brilliant Blue or specific protein stains.
2. Two-dimensional BN-SDS-PAGE
Two-dimensional BN-SDS-PAGE adds a second dimension to BN-PAGE for analyzing the components of protein complexes through SDS-PAGE.
Second Dimension Materials and Solutions
- SDS-PAGE Loading Buffer: Contains SDS and DTT, used for separating the components of protein complexes in the second dimension.
Steps:
1. First Dimension BN-PAGE: Perform as described in the BN-PAGE steps above.
2. Prepare Second Dimension: Cut the gel strip from the first dimension electrophoresis and incubate it in a loading buffer containing SDS to allow SDS to permeate the gel and bind with the proteins.
3. Second Dimension SDS-PAGE: Place the treated gel strip on the SDS-PAGE gel and perform second-dimension electrophoresis. This step further separates the complex components from the first dimension based on protein size.
4. Staining/Detection: Stain and detect the gel using Coomassie Brilliant Blue or silver stain as in BN-PAGE.
BiotechPack, A Biopharmaceutical Characterization and Multi-Omics Mass Spectrometry (MS) Services Provider
Related Services:
2D Blue Native/SDS-PAGE Complex Analysis Services
SDS-PAGE Protein Purity Analysis
Characterization of Protein Purity and Homogeneity
Protein Purity Analysis (Size Exclusion/Reverse Phase Chromatography)
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