Definition of Gel Electrophoresis:

Gel Electrophoresis is the process in which molecules (such as proteins, DNA, or RNA fragments) can be separated according to size and electrical charge by applying an electric current to them. The current forces the molecules through pores in a thin layer of gel, a jelly-like substance. The gel can be made so that its pores are just the right dimensions for separating molecules within a specific range of sizes and shapes. Smaller fragments usually travel further than large ones. The process is sometimes called gel electrophoresis.

Gel Electrophoresis is electrophoresis performed in silica gel, a porous inert medium. Electrophoresis is a process in which colloidal particles or macromolecules (nucleic acids or proteins) with a net electric charge migrate in a solution under the influence of an electric current (also known as cataphoresis or kataphoresis). Electro refers to the energy of electricity, and phoresis derives from the Greek verb phoros, meaning ‘to carry across’.

Separation of macromolecules (or large molecules) depends upon two forces, namely mass and charge.

Principles of Gel Electrophoresis

Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely- used techniques in biochemistry and molecular biology. When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their chargeProteins and nucleic acids are electrophoresed within a "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.

The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks:

Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic.

Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders).

Polyacrylamide gels have a rather small range of separation, but very high resolving power. TYPES OF ELECTROPHORESIS

One-Dimensional

1.Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

SDS-PAGE is a common form of electrophoresis for analyzing proteins. The SDS part of the name is a protein denaturing detergent that causes the molecule to unfold. The protein polypeptides move through the gel at different rates depending on mass, allowing researchers to study proteins based on size.

2.Isoelectric Focusing(IEF)

In Isoelectric focusing, proteins are separated by electrophoresis in a pH gradient based on their isoelectric point(pl). At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will move towards the more negative end of the gel and if they are negatively charged they will move towards the more positive end of the gel. At its isoelectric point, since the protein molecule carry no net charge it accumulates or focuses into a sharp band.

3.Native-PAGE

Native PAGE is used to separate proteins in their native states according to difference in their charge density. It is used for prepartion of purified and active proteins. In native PAGE the mobility depends on both the protein's charge and its hydrodynamic size. Native PAGE can be carried out near neutral pH to avoid acid or alkaline .The apparatus is kept cool to minimize denaturation of proteins and proteolysis.

Two Dimensional Polyacrylamide gel Electrophoresis(2D-PAGE) 2D Electrophoresis is a technique efficient separation technique for proteins. This technique results in gels that contain spots. Each spot on the gel corresponds to a different protein. Then the gels are stained similarly as to 1D analysis. 2D Electrophoresis is widely used, and certain methods of it can be coupled with mass spectrometry in order to identify proteins.

Differential in Gel Electrophoresis(DIGE)

Differential in Gel Electrophoresis (DIGE) is a technique to monitor the differences in proteomic profile between cells in different functional states. This technology allows for simultaneous separation and comparison of up to three samples on one gel

QPNC-PAGE

Quantitative Preparative Native Continuous PolyAcrylamide GelElectrophoresis is a technique to isolate active or native metalloproteins (e.g., metal chaperones, prions, metal transport proteins, amyloids, metalloenzymes, metallopeptides) in biological samples and to resolve properly and improperly folded metal cofactor-containing proteins in complex protein mixtures

Capillary

Capillary electrophoresis is a method similar to SDS-PAGE. It separates molecules based on their charge and mass. Molecules are placed in rows called capillaries filled with conductive, electrolyte fluid. This method is an older technique introduced in the 1960s.

Applications of Electrophoresis

1. DNA Analysis

Electrophoresis is one way of analyzing DNA, which is the code that contains all the traits you inherited from your parents. DNA is arranged in sequences, one sequence represents the color of your eyes and another sequence represents the color of your skin. Through electrophoresis, specific DNA sequences can be analyzed, isolated and cloned

2. Antibiotics Analysis

The application of electrophoresis in antibiotic studies dates back to the 1950s. Further studies led to improved electrophoretic techniques and new antibiotics. These drugs, such as penicillin, are among the widely prescribed drugs against bacterial infections. With electrophoresis, experts are not only able to synthesize new antibiotics but are also able to analyze which types of bacteria are antibiotic-resistant.

3. Vaccine Analysis

Vaccine analysis is one of the many important applications of electrophoresis. There are several vaccines that have been purified, processed and analyzed through electrophoresis, such as the influenza vaccine, hepatitis vaccine and polio vaccine. The exact steps done in the vaccine analysis, however, cannot be determined due to confidentiality reasons of the pharmaceutical companies. Nevertheless, data reports from vaccine manufacturers such as Wyeth, Merck and Sanofi-Aventis presents electrophoresis as an effective vaccine analysis method.

References

• Berg,Tymoczko,Stryer.Biochemistry:Fifth Edition.New York, New York.W.H.Freeman and Company,2002. • http://www.chemsoc.org/ExemplarChem/entries/2003/leeds_chromatog raphy/chromatography/