2D Blue Native/SDS-PAGE Protein Complex Analysis Service

In biological membranes, many proteins form complexes. MtoZ Biolabs provides comprehensive subunit analysis of these protein complexes using 2D blue native (BN)/SDS-PAGE. In 2D BN/SDS-PAGE analysis, samples undergo 1D separation through BN-PAGE, followed by 2D separation via SDS-PAGE.

 

Protein complexes organize and maintain cellular and organelle functions at all levels of complexity in both time and space, including cell development and division, transcription and translation, respiration and photosynthesis, transport, and metabolism. Protein complexes may exhibit partial and transient changes and show varying stability, making their identification and analysis challenging. BN-PAGE is a high-resolution separation technique for protein complexes. It can be used to determine the size, relative abundance, and subunit composition of protein complexes.

 

In 2D BN-PAGE analysis, protein or protein complex samples are separated in 1D BN-PAGE under native conditions. Coomassie brilliant blue dye carries a negative charge and binds non-specifically to all proteins. Unlike detergents, coomassie brilliant blue preserves the structure of protein complexes. Its binding also reduces the tendency for protein aggregation during electrophoresis. The electrophoretic mobility of the samples depends on the negative charge of the bound coomassie brilliant blue as well as the size and shape of the complex. After separation by BN-PAGE, protein and protein complex samples are denatured in-gel with SDS and then separated using 2D SDS-PAGE.

 

The combination of BN-PAGE and SDS-PAGE enables monomeric proteins to migrate along a hyperbolic diagonal due to the gradient gel in 1D and linear gel in 2D. Components of protein complexes appear below this diagonal. Proteins representing the subunits of the same protein complex can be found in a vertical line in 2D, while multiple spots of the same protein along the horizontal line indicate that the protein is simultaneously present in several different protein complexes.


2D BN/SDS-PAGE analysis provides valuable information regarding the size, quantity, protein composition, stoichiometry, and relative abundance of samples, making it a powerful tool for studying protein complexes.