In biological membranes, many proteins are organized in complexes. Creative Proteomics offers a method for the global analysis of the subunits of these protein complexes through 2D Blue Native / SDS-PAGE analysis. In 2D BN / SDS-PAGE analysis, the samples were analyzed in the 1st dimension by blue native polyacrylamide gel electrophoresis (BN-PAGE), and then separated by SDS-PAGE in the 2nd dimension which is 90 degrees from the first.
BN-PAGE provides the technology for high resolution separation of protein complexes. The blue native electophoresis protocol is used to determine the size, relative abundance and subunit composition of protein complexes. Protein complexes organize and maintain the cellular and organelle functions on all levels of complexity in time and space, including cell development and division, transcription and translation, respiration and photosynthesis, transport and metabolism. Protein complexes may exhibit partial and temporal changes, as well as exhibit different stabilities, making the identification and analysis extremely difficult.
In 2D BN/SDS-PAGE analysis, the protein or protein complex samples are separated under native conditions in a first-dimension BN-PAGE. The Coomassie blue dye, which is negatively charged and binds nonspecifically to all the proteins, is used in BN-PAGE. Coomassie blue does not act as a detergent, and it preserves the structure of protein complexes. In addition, the binding of Coomassie blue decreases the tendency of proteins to aggregate during the stacking step of the electrophoresis process. Therefore, the electrophoretic mobility of the samples is determined by the negative charge of the bound Coomassie blue and the size and shape of the complex.
For 2D BN/SDS-PAGE, the proteins and protein complex samples are denatured by SDS in the gel strip after the separation by BN-PAGE before they are applied to a second-dimension SDS-PAGE gel.
By combination of first dimension BN-PAGE and second dimension SDS-PAGE, monomeric proteins will migrate in a hyperbolic diagonal due to the gradient gel in the first and a linear gel in the second dimension, while components of protein complex are located below this diagonal. Proteins that represent subunits of the same protein complex can be found in one vertical line in the second dimension, whereas several spots of the same protein in a horizontal line indicate the presence of the protein in several distinct protein complexes.
2D BN/SDS-PAGE can provide information about the size, number, protein composition, stoichiometry and relative abundance of the sample, and has been proved to be a useful tool for the study of protein complex.