Two dimensional gel electrophoresis, commonly abbreviated as 2-DE or 2D-PAGE, is commonly used to separate proteins. The two dimensions that proteins are separated into in this technique correspond to isoelectric point and molecular mass.
The protein separation of the first dimension is based on different protein charges. The state of charge of a protein is dependent on the pH and the amino-acid composition. During isoelectric focussing, pH-gradients are created in the matrix in which each single polypeptide of a protein mixture is trapped at the pH of their isoelectric point. The pI-value of protein defines the pH-value, at which positive and negative charges of amino-acids are compensated. At this point, the proteins are electrically neutral and do not migrate any more in an electric field. The pH-gradient is created by a mixture of ampholytes (polymers with attached amino- and carboxyl groups).
Before separating the proteins by mass, they are treated with sodium dodecyl sulfate (SDS). This denatures the proteins (it unfolds them into long, straight molecules) and attaches a number of SDS molecules roughly proportional to the protein's length. Because a protein's length (when unfolded) is roughly proportional to its mass, this is equivalent to saying that it attaches a number of SDS molecules roughly proportional to the protein's mass. Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass-to-charge ratio as each other. Next, an electric potential is again applied, but at a 90 degree angle from the first field. The proteins will be attracted to the more negative side of the gel proportionally to their mass-to-charge ratio. The proteins' progress will be slowed by frictional forces. This frictional slowing is roughly inversely proportional to the protein's size. The electric field is applied for as long as it takes the smallest protein to reach the far end of the gel.
