Elution of proteins from a gel is a method of obtaining separated or purified proteins for other assays such as sequencing and tryptic digestion. One percent agarose gels can be used to separate molecules greater than 200,000 Daltons. IEF is usually performed on horizontal gels which allow efficient cooling and easy sample application. These ampholytes have a wide range of isoelectric points in which the pH steadily increases from the anode to the cathode. Resolution of proteins by isoelectric focusing is due to the pH gradient, the thickness of the gel, the time of electrophoresis, the applied voltage, and the diffusion of protein into the gel.
Resolution is the separation of protein bands relative to their band widths.
It is denoted by the differences in IpH between two distinguishable bands. A narrow pH range means more distance between IpH's and better resolution. High electrical fields produce shorter run times, but large amounts of heat can cause the gels to burn. Resolution decreases with increasing temperature due to increased diffusion. Resolution can also decrease with excessive time, due to gradient drift.