2D Gel Electrophoresis
 
Techniques
  
 
 
 
2D electrophoresis using immobilized pH gradients and SDS-PAGE
Figure 1 - 2D electrophoresis (click to enlarge).

In 2D electrophoresis (2DE) proteins are first separated according to their iso-electric points (1st dimension) and subsequently according to their molecular weights (second dimension). Separation in two different dimensions (pI and Mw) makes 2DE capable of separating hundreds to thousands of proteins at high resolution.

Iso-electric focusing (IEF) of proteins on their pI in the first dimension was first performed using carrier ampholites (CA) but was later on replaced by the use of IPG strips. This introduction of IPG strips for isoelectric focusing has been a significant milestone in the field of electrophoresis. Unlike conventional IEF with CA-formed pH gradients, IPG gels contain chemically immobilized buffering and titrant groups that cannot migrate in the electric field. This permits steady state focusing, eliminating the problem of cathodic drift observed in conventional IEF and thereby establishes highly reproducible protein patterns.
Use of IPG strips has also improved the protein load capacity needed for the analysis of low abundance proteins in proteomics. Use of wide range pH IPG strips offer an overview of the state of the proteome whereas narrow range pH IPG strips offer high-resolution separation for the detection of a maximum number of spots.
 
BlueNative 2D electrophoresis
Figure 2 - BN 2D electrophoresis (click to enlarge).
Conventional 2DE using IEF as first dimension and subsequently SDS-PAGE as second dimension offers great possibilities for the analysis of denatured proteins. But as a result of the denaturing method no information about the organization of protein complexes or protein-protein interactions can be obtained. Also the analysis of membrane proteins, hydrophobic, alkalic, and high molecular weight proteins can often be troublesome. A promising electrophoresis method, which offers high-resolution analysis of native protein complexes and hydrophobic proteins, is Blue-Native (BN) electrophoresis. Originally developed by Schägger and Jagow in 1991 to study membrane-bound respiratory chain complexes (e.g. NADH ubiquinone oxidoreductase or complex I) it is now widely used as an one dimensional electrophoresis method or as first dimensional separation in two- or three-dimensional electrophoresis. In 2D BN-PAGE (figure 2) complexes are first separated in their native form by BN electrophoresis. This is then followed by the denaturation of proteins and subsequent transfer of the BN gel strip to a second dimension SDS polyacrylamide gel. Dissociated complex subunits are then separated by SDS-PAGE in a second electrophoresis step.

Blue-Native electrophoresis owes its name to the blue color during electrophoresis caused by the crucial compound Serva Blue G (coomassie dye). The basic principles of BN comprises the use of mild neutral detergents for protein solubilization and the use of coomassie blue G 250 to give a charge to proteins and complexes. The coomassie dye binds to the surface of all hydrophobic proteins but not to all water-soluble proteins. The binding of anionic dye molecules results in the following crucial effects: (1) The binding of coomassie dye shifts the iso-electric points of proteins to the negative which allows migration to the anode at pH7.5. Migrating proteins are then separated by their specific masses but not according to their charge/mass ratio: The decreasing pore size of the polyacrylamide-gradient gel (which is used in the first dimension) leads to a mass-dependent reduction of the protein migration velocity and to an almost complete stop at a mass-specific pore size limit . (2) Hydrophobic protein aggregation is considerably reduced by repelling negative charges located on the surface of individual proteins. (3) Coomassie bound hydrophobic proteins are water-soluble and there is therefore no need for including detergent in the gel. This greatly reduces the risk of denaturing detergent-sensitive proteins. (4) Recovery of native proteins by electroelution is facilitated by the fact that native proteins are visible as migrating blue bands during electrophoresis.