Isoelectric Focusing

Gel Electrophoresis of Proteins

| National Diagnostics | Electrophoresis Articles, Gel Electrophoresis of Proteins

Conventional electrophoresis techniques can separate up to 100 different proteins on one run. Typically, cell or tissue extracts contain thousands of proteins, most of which will not be resolved into single bands using a separation based on any one parameter, such as size or net charge. For any one size range, there is a high probability of more than one protein (out of thousands) falling into this range. Separation on the basis of two parameters, usually size and isoelectric point, lowers the probability that two proteins will overlap, and allows the resolution of thousands of protein species on one gel. Such two dimensional separations are carried out by running a one dimensional gel to separate by the first parameter, and then laying this gel or a lane cut from it, across the top of the second dimension gel, the first gel serving as the "sample" for the second. Typically, the second dimension gel is an SDS PAGE gel, because this gives a direct measurement of a protein's size. The first dimension usually employs isoelectric focusing (IEP) as the means of separation. IEF gives a measurement of the isoelectric point (pI) of the protein - the pH at which the protein sample will not migrate in an electric field, the protein's net charge being zero. A two dimensional gel can give size and pI information on thousands of proteins in one run.

Further Exploration on Isoelectric Focusing

Isoelectric focusing employs a pH gradient extending the length of an electrophoresis gel. A protein stops migrating when it enters the zone in which the surrounding pH equals its isoelectric point, pI. At any other point in the gradient, the protein acquires a charge which causes it to migrate toward its pI (green and blue arrows).

Proteins carry charged groups on their surface. Each of these functional groups has a pK, which corresponds to the pH at which half of the members of that group are protonated. Above the pK that group can be considered fully protonated, below the pK, fully deprotonated. Thus, as the pH changes, the net charge on a protein's surface will change. At high pH, most proteins will have many deprotonated surface groups and will carry a net negative charge. At low pH, with many protons added to the surface, most proteins have a net positive charge. At some intermediate pH, different for every protein, the net charge on the protein will be zero. It is important to remember that this is a net charge - the protein is not uncharged - it carries equal numbers of positive and negative charges. The pH at which a protein has a net charge of zero is designated as its isoelectric point (pI).

A protein dissolved in buffer at its pI has no net charge and thus no net electrophoretic mobility. In isoelectric focusing (IEF) a pH gradient is established along the length of the gel. Proteins migrate through this gradient until they reach their pI. The gradient is set up so that negatively charged molecules migrate into a decreasing pH. If a protein is in a region where the pH is above its pI, it has a negative charge and moves to a lower pH. If it is in a pH below its pI, it has a positive charge which moves it into higher pH regions. This gives rise to the "focusing" aspect of IEF, as proteins are continually swept back into tight bands centered on the appropriate pI (see figure below). IEF is thus an equilibrium electrophoresis system, runs until protein movement ceases.

Ampholytes

The pH gradient in IEF gels is generated by the inclusion of ampholytes, low molecular weight amphoteric molecules. A mixture of ampholytes is used, each having a different pI. Like protein molecules, the ampholytes migrate through the gel until they reach a region where the pH is equal to their pI. Unlike the proteins, the ampholytes are present in high enough concentration to change their local pH. The gel is set up with a uniform mixture of ampholytes throughout, and its anodic and cathodic ends are immersed in dilute acid and base respectively. Ampholytes near the ends of the gels will be positively charged near the positive electrode, and negatively charged near the negative electrode. They, therefore, begin to migrate into the gel, with the most charged (ie the ones furthest from their pI) moving the fastest. Over time they separate into zones of defined pH. If the ampholyte system is well designed, a smooth gradient of pH is created, with no abrupt changes, or "steps." Commercial systems are available in a broad range (2 - 12 pH) or narrow range (extending 2 pH units across the gel).

Various mixtures of amphoteric substances have been used as ampholytes: amino acids, proteins, and poly acidic polybasic synthetic molecules. Natural amino acids have poor conductivity and poor buffering capacity in their zwitterionic state, making them poor candidates. Proteins can be good ampholytes, but they interfere with the analysis of the sample, by introducing new proteins into the mixture. Polycarboxylic acid polyamines are the most commonly used ampholytes. These molecules have excellent buffering capacity and conductivity across a broad pH range and are usually provided in a molecular weight range of 300 - 500, which is small enough to avoid interference with most subsequent processing. Their sole disadvantage is that they may bind tightly to the proteins, due to ionic interactions, and can be very difficult to remove.