Electrophoresis

 Electrophoresis is the motion ofdispersed particles relative to a fluid under the influence of a spatially uniformelectric field. [1][2][3][4][5][6] This electrokinetic phenomenon was observed for the first time in 1807 by Ferdinand Frederic Reuss (Moscow State University),[7] who noticed that the application of a constant electric field causedclayparticles dispersed in water to migrate. It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid. It is the basis for a number of analytical techniques used in biochemistry for separating molecules by size, charge, or binding affinity.

Electrophoresis of positively charged particles (cations) is called cataphoresis, while electrophoresis of negatively charged particles (anions) is called anaphoresis. Electrophoresis is a technique used in laboratories in order to separate macromolecules based on size. The technique applies a negative charge so proteins move towards a positive charge. This is used for both DNA and RNA analysis. Polyacrylamide gel electrophoresis (PAGE) has a clearer resolution than agarose and is more suitable for quantitative analysis. In this technique DNA foot-printing can identify how proteins bind to DNA. It can be used to separate proteins by size, density and purity. It can also be used for plasmid analysis, which develops our understanding of bacteria becoming resistant to antibiotics.

Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA andproteins) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge and/or size (IEF agarose, essentially size independent) and inbiochemistryand molecular biologyto separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteinsby charge. Nucleic acid molecules are separated by applying an electric fieldto move the negatively charged molecules through a matrix of agarose or other substances. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving.[2]Proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins. Gel electrophoresis can also be used for separation of nanoparticles.

Gel electrophoresis uses a gel as an anticonvective medium and/or sieving medium during electrophoresis, the movement of a charged particle in an electrical field. Gels suppress the thermal convection caused by application of the electric field, and can also act as a sieving medium, retarding the passage of molecules; gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied.[3] DNA Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, orSouthern blotting for further characterization. 

1.  Physical basis 

In simple terms, electrophoresis is a process which enables the sorting of molecules based on size. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agarorpolyacrylamide. The electric field consists of a negative charge at one end which pushes the molecules through the gel, and a positive charge at the other end that pulls the molecules through the gel. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel.

The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separatingproteins or small nucleic acids (DNA, RNA, oroligonucleotides) the gel is usually composed of different concentrations of acrylamide and a crosslinker, producing different sized mesh networks ofpolyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purifiedagarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast topolyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Agarose is composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel with large pores allowing for the separation of macromolecules

"Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass when the charge to mass ratio (Z) of all species is uniform. However when charges are not all uniform then, the electrical field generated by the electrophoresis procedure will affect the species that have different charges and therefore will attract the species according to their charges being the opposite. Species that are positively charged (cations) will migrate towards the cathode which is negatively charged. If the species are negatively charged (anions) they will migrate towards the positively charged anode.

If several samples have been loaded into adjacent wells in the gel, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components. Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

There are limits to electrophoretic techniques. Since passing current through a gel causes heating, gels may melt during electrophoresis. Electrophoresis is performed in buffer solutions to reduce pH changes due to the electric field, which is important because the charge of DNA and RNA depends on pH, but running for too long can exhaust the buffering capacity of the solution. Further, different preparations of genetic material may not migrate consistently with each other, for morphological or other reasons.

2. Gel conditions 

Denaturing 

Denaturinggels are run under conditions that disrupt the natural structure of the analyte, causing it to unfold into a linear chain. Thus, the mobility of each macromoleculedepends only on its linear length and its mass-to-charge ratio. Thus, the secondary, tertiary, and quaternary levels ofbiomolecular structure are disrupted, leaving only the primary structure to be analyzed. Nucleic acids are often denatured by includingurea in the buffer, while proteins are denatured using sodium dodecyl sulfate, usually as part of the SDSPAGE process. For full denaturation of proteins, it is also necessary to reduce the covalent disulfide bonds that stabilize their tertiaryandquaternary structure, a method called reducing PAGE. Reducing conditions are usually maintained by the addition ofbeta-mercaptoethanol ordithiothreitol. For general analysis of protein samples, reducing PAGE is the most common form of protein electrophoresis.

Denaturing conditions are necessary for proper estimation of molecular weight of RNA. RNA is able to form more intramolecular interactions than DNA which may result in change of itselectrophoretic mobility. Urea, DMSO andglyoxal are the most often used denaturing agents to disrupt RNA structure. Originally, highly toxic methylmercuryhydroxide was often used in denaturing RNA electrophoresis,[12] but it may be method of choice for some samples.

Denaturing gel electrophoresis is used in the DNA and RNA banding patternbased methods DGGE (denaturing gradient gel electrophoresis),[14] TGGE(temperature gradient gel electrophoresis), and TTGE (temporal temperature gradient electrophoresis).

Native gels are run in non-denaturing conditions, so that the analyte's natural structure is maintained. This allows the physical size of the folded or assembled complex to affect the mobility, allowing for analysis of all four levels of the biomolecular structure. For biological samples, detergents are used only to the extent that they are necessary tolyse lipid membranes in the cell. Complexes remain—for the most part—associated and folded as they would be in the cell. One downside, however, is that complexes may not separate cleanly or predictably, as it is difficult to predict how the molecule's shape and size will affect its mobility.

Unlike denaturing methods, native gel electrophoresis does not use a chargeddenaturingagent. The molecules being separated (usuallyproteinsornucleic acids) therefore differ not only in molecular mass and intrinsic charge, but also the cross-sectional area, and thus experience different electrophoretic forces dependent on the shape of the overall structure. For proteins, since they remain in the native state they may be visualised not only by general protein staining reagents but also by specific enzyme-linked staining. 

Buffers 

Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value. There are a number of buffers used for electrophoresis. The most common being, for nucleic acids Tris/Acetate/EDTA(TAE), Tris/Borate/EDTA (TBE). Many other buffers have been proposed, e.g. lithium borate, which is almost never used, based on Pubmed citations (LB), iso electric histidine, pK matched goods buffers, etc.; in most cases the purported rationale is lower current (less heat) and or matched ion mobilities, which leads to longer buffer life. Borate is problematic; Borate can polymerize, and/or interact with cis diols such as those found in RNA. TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product. LB is relatively new and is ineffective in resolving fragments larger than 5 kbp; However, with its low conductivity, a much higher voltage could be used (up to 35 V/cm), which means a shorter analysis time for routine electrophoresis. As low as one base pair size difference could be resolved in 3% agarose gel with an extremely low conductivity medium (1 mM Lithium borate).

Most SDS-PAGE protein separations are performed using a "discontinuous" (or DISC) buffer system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band in a process called isotachophoresis. Separation of the proteins by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of the proteins. 

3. Visualization 

After the electrophoresis is complete, the molecules in the gel can be stainedto make them visible. DNA may be visualized using ethidium bromide which, when intercalated into DNA, fluoresce underultraviolet light, while protein may be visualised using silver stain or Coomassie Brilliant Bluedye. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the molecules to be separated contain radioactivity, for example in a DNA sequencinggel, anautoradiogram can be recorded of the gel. Photographs can be taken of gels, often using a Gel Doc system. 

4. Application 

 Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. inrestriction mappingof cloned DNA. 

 Analysis of PCR products, e.g. in molecular genetic diagnosisor genetic fingerprinting 

 Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior to Northern transfer.