Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss (Moscow State University), who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid. Electrophoresis of positively charged particles (cations) is called cataphoresis, while electrophoresis of negatively charged particles (anions) is called anaphoresis.
Gel electrophoresis is a process to separate strains of DNA by pushing it through a gel placed in a heated (and covered) table. The porousness of the gel causes larger fragments to separate first while smaller fragments continue through the gel. Since its first experimental use in the 1930's, gel electrophoresis has been refined through the use of different types of gel. Beginning with sucrose and then starch gels, DNA separation in electrophoresis greatly increased with modern use of agarose and acrylaminde gels. With the development of capillary electrophoresis, certain advantages and disadvantages have become clear for both methods.
The theory of electrophoresis:
The concept here is simple enough. Like centrifugation, the molecules feel a force pushing them in one direction. However, in this case, the force involved is due to the electric field acting on the charge of the molecule and is given by F = EQ where F is the force, E is the electric field and Q is the charge. Obviously the greater the charge on the molecule, the greater the force. Thus, for two molecules of the same size, the one with the larger charge will move faster in the electric field. Now, it is less obvious that molecules with a larger mass will move more slowly. This actually comes about because the frictional forces that slow a molecule traveling through solution down depend on the molecules size. The speed at which molecules go through a solution is determined by the point at which the forces driving it forward are just balanced by the frictional forces generated by the motion. The greater the mass of the molecule, the greater the size, in general, and therefore the friction the molecule will generate when traveling through the solution. It turns out that in fact the electrophoretic mobility of a molecule depends on its charge to mass ratio. Two different sized molecules with the same charge to mass ratio should run with the same mobility in a uniform electric field and a perfect world.
• A technique whereby charged molecules are separated by the use of an electric field.
• During electrophoresis, charged molecules will migrate towards an opposite charge.
• A mixture of molecules of various sizes will migrate at different velocities and will be separated.
• Electrophoresis is usually carried out in an aqueous solution.
• Electrophoresis is a commonly used technique in many scientific fields.
Types of electrophoresis:
There are quite a number of types of electrophoresis commonly used. It is not possible to go through them all in any detail here, but a brief description of a few of the most common types follows:
SDS-PAGE: One of the most common means of analyzing proteins by electrophoresis is by using Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis. SDS is a detergent which denatures proteins by binding to the hydrophobic regions and essentially coating the linear protein sequence with a set of SDS molecules. The SDS is negatively charged and thus becomes the dominant charge of the complex. The number of SDS molecules that bind is simply proportional to the size of the protein. Therefore the charge to mass ratio should not change with size. In solution (water), in principle all different sized proteins covered with SDS would run at about the same mobility. However, the proteins are not run through water. Instead they are run through an inert polymer, polyacrylamide. The density and pore size of this polymer can be varied by just how you make it (concentration of monomer and of cross-linking agent). Thus, the size of molecules that can pass through the matrix can be varied. This determines in what molecular weight range the gel will have the highest resolving power.
Native Gels: It is also possible to run protein gels without the SDS. These are called native gels in that one does not purposely denature the protein. Here, the native charge on the protein (divided by its mass) determines how fast the protein will travel and in what direction.
Electrofocusing Gels: Another variation of gel electrophoresis is to pour a gel that purposely has a pH gradient from one end to the other. As the protein travels through this pH gradient, its various ionizable groups with either pick up or lose protons. Eventually, it will find a pH where its charge is zero and it will get stuck (focused) at that point.
DNA Agarose Gels: A simple way of separating fairly large fragments of DNA from one another by size is to use an agarose gel. Agarose is another type of matrix used for many purposes (such as the support for the growth of bacteria on plates). DNA does not need a detergent, since it already has a large under of negative phosphate groups evenly spaced. Thus, as with SDS-PAGE, the charge to mass ratio is constant. Also like SDS-PAGE, the separation results from the matrix itself. The range of size sensitivity can be varied by changing the density of the agarose.
DNA denaturing polyacrylamide gels (often called sequencing gels): To look at smaller DNA molecules with much higher resolution, people generally denature the DNA via heat and run it through a thin polyacrylamide gel that is also kept near the denaturing temperature. These gels usually contain additional denaturing compounds such as Urea. Two pieces of DNA that differ in size by 1 base can be distinguished from each other this way.
Capillary electrophoresis: It has become popular to separate molecules electrophoretically by running them into and through a capillary tube. This is fast and accurate, but does not allow much sample to be loaded on the gel at once.
About Author / Additional Info:
Trending Articles ( Receiving maximum views in the last few days )