Isoelectric point is the point at which the overall charge of the protein is zero (a neutral charge). Separation of proteins at the isoelectric point is called isoelectric focusing. In isoelectric focusing a gradient of pH and an electric potential are applied across the gel, making one end more positive than the other. Separation occurs on the basis of the positive or negative groups present on the molecule. If they are positively charged, they will be pulled toward the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will migrate through the gel and will accumulate at their isoelectric point. At this stage the protein net charge is zero and therefore does not move in an electric field.