2D Blue Native/SDS-PAGE Complex Analysis: Requesting a Protocol

2D Blue Native/SDS-PAGE is a widely used technique for analyzing protein complexes and their subunit composition. The following describes the standard experimental procedure for this method.

 

1. Sample Preparation

Extract protein samples and stabilize protein complexes using an appropriate buffer (e.g., containing an appropriate concentration of Digitonin or Triton X-100).

 

2. First Dimension: Blue Native PAGE

• Prepare Blue Native PAGE gels (typically 4–16% gradient gels).
• Mix protein samples with Coomassie Brilliant Blue G-250 dye, ensuring that buffers free of sulfate are used.
• Load the samples and perform electrophoresis at 4 °C using suitable cathode and anode buffers.

 

3. Gel Strip Cutting and Treatment

• After the first-dimension electrophoresis, excise gel strips containing protein bands along the longitudinal axis of the gel.
• Incubate the strips in an SDS-containing buffer with gentle agitation to facilitate SDS penetration.

 

4. Second Dimension: SDS-PAGE

• Place the prepared gel strips onto an SDS-PAGE gel (commonly 12–20% gradient gels).
• Conduct SDS-PAGE to resolve the subunits of the protein complexes.

 

5. Protein Staining and Image Analysis

• Visualize proteins on the SDS-PAGE gels using Coomassie Brilliant Blue or silver staining.
• Analyze protein spots with image analysis software to determine the subunit composition of the complexes.

 

6. Data Interpretation

Based on the electrophoretic mobility and molecular size of the protein spots, deduce the potential subunit composition of the protein complexes.

 

Adjustments to the procedure may be made according to specific experimental requirements and protein characteristics.

 

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