Workflow of 2D Blue Native/SDS-PAGE Protein Complex Analysis

2D Blue Native/SDS-PAGE complex analysis is a robust technique for separating and analyzing protein complexes. It combines Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) with subsequent SDS-PAGE, providing detailed information about the native state and components of protein complexes in the same gel. This method is invaluable for elucidating protein interactions, complex structures, and functions.

 

Materials and Reagents

1. Buffers and Reagents

(1) Blue Native Electrophoresis Buffer

(2) SDS-PAGE Buffer

(3) Sample Buffer (containing Coomassie Brilliant Blue G-250)

(4) SDS-PAGE Sample Buffer

(5) Electrophoresis Buffer

 

2. Equipment

(1) Electrophoresis Apparatus

(2) Polyacrylamide Gels

(3) Power Supply

 

Experimental Steps

1. Sample Preparation

(1) Extract protein samples and measure their concentration.

(2) Combine protein samples with the sample buffer, ensuring the final concentration of Coomassie Brilliant Blue G-250 is approximately 0.02%.

 

2. Blue Native PAGE (BN-PAGE)

(1) Prepare polyacrylamide gels with an appropriate concentration, typically 4-16% gradient gels.

(2) Apply the samples to the BN-PAGE gel.

(3) Perform electrophoresis at 4°C, starting at a low voltage (e.g., 50V) until the samples enter the gel, then increasing to a higher voltage (e.g., 100-200V) for separation.

 

3. Gel Processing

After completing BN-PAGE, cut the gel into strips and incubate them in SDS-containing buffer to remove Coomassie Brilliant Blue G-250 and denature the proteins.

 

4. SDS-PAGE

(1) Prepare the second-dimension SDS-PAGE gel, typically a 10-15% polyacrylamide gel.

(2) Place the treated BN-PAGE strips at the top of the SDS-PAGE gel.

(3) Conduct SDS-PAGE at a constant voltage (e.g., 150V) until the samples are fully separated.

 

5. Protein Detection

(1) Stain the SDS-PAGE gel using suitable methods (e.g., silver staining or Coomassie Brilliant Blue staining) to visualize the separated protein bands.

(2) Document and analyze the images of the protein bands.

 

Results Analysis

2D Blue Native/SDS-PAGE complex analysis provides a comprehensive profile of protein complexes. BN-PAGE, as the first dimension, reveals the native state of protein complexes, while SDS-PAGE, as the second dimension, provides detailed information about the individual components of each complex. Combining these two dimensions allows for an in-depth investigation into the structure and function of protein complexes.

 

The 2D Blue Native/SDS-PAGE complex analysis technique is a powerful and widely used tool that offers extensive data for the study of protein complexes. Through meticulous sample preparation, precise electrophoresis, and thorough result analysis, researchers can gain profound insights into the dynamic changes and functional mechanisms of protein complexes.