Proteomics/Protein Separations- Electrophoresis

Definitions
e•lec•tro•pho•re•sis (ĭ-lĕk'trō-fə-rē'sĭs) n. 1) The migration of charged colloidal particles or molecules through a solution under the influence of an applied electric field usually provided by immersed electrodes. Also called cataphoresis. 2) A method of separating substances, especially proteins, and analyzing molecular structure based on the rate of movement of each component in a colloidal suspension while under the influence of an electric field.

an•a•lyte (a-nə-līt) n. A chemical substance that is the subject of chemical analysis.



Electrophoresis Theory
Separation by electrophoresis depends on differences in the migration velocity of ions or solutes through a given medium in an electric field. The electrophoretic migration velocity of an analyte is:

$$v_p = \mu_p E$$

where E is the electric field strength and $$\mu_p$$ is the electrophoretic mobility.

The electrophoretic mobility is inversely proportional to frictional forces in the buffer, and directly proportional to the ionic charge of the analyte. The forces of friction against an analyte are dependent on the analyte's size and the viscosity (η) of the medium. Analytes with different frictional forces or different charges will separate from one another when they move through a buffer. At a given pH, the electrophoretic mobility of an analyte is:

$$\mu_p = \frac{z}{6\pi \eta r}$$ where r is the radius of the analyte and z is the net charge of the analyte.

Differences in the charge to size ratio of analytes causes differences in electrophoretic mobility. Small, highly charged analytes have greater mobility, whereas large, less charged analytes have lower mobility. Anionic surfactants are often used to denature proteins before electrophoresis. This gives all proteins approximately the same charge. Since their charges are all equal their mobility is now a function of only their masses. Electrophoretic mobility is an indication of an analyte's migration velocity in a given medium. The net force acting on an analyte is the balance of two forces: the electrical force acting in favor of motion, and the frictional force acting against motion. These two forces remain steady during electrophoresis. Therefore, electrophoretic mobility is a constant for a given analyte under a given set of conditions.

Applications of Electrophoresis
Electrophoresis has a wide variety of applications in proteomics, forensics, molecular biology, genetics, biochemistry, and microbiology.

One of the most common uses of electrophoresis is to analyze differential expression of genes. Healthy and diseased cells can be identified by differences in the electrophoretic patterns of their proteins. Proteins themselves can also be characterized in this way, and some sense of their structure can be derived from the masses of fragments inside the gel.

There are many different types of electrophoresis, and each can be used for something different. Two-dimensional (2-D) electrophoresis, for example, has the ability to discern many more proteins than most of its contemporaries. Many of these methods will be discussed in detail throughout this chapter.