Explaining the Process of Gel Electrophoresis

Arne Tiselius, in the 1930s, was the first to develop the basic principle underlying electrophoresis with the help of a rough model, but the actual modern-day technique was developed in the 1950s by Oliver Smithies.

Gel electrophoresis is a technique used in clinical as well as laboratory settings, to separate mixed populations of macro-molecules. Nucleic acids (DNA and RNA) are separated based on their fragment length and the inherent charge of the molecules. Proteins are separated based on their charge and size.

The gel utilized for the separation of these molecules acts as an anti-convective, sieving medium that allows the movement of a charged particle in an electric field. Due to this property, this technique can also be used for separation of nanoparticles.

The gel maintains the finished separation of the molecules, hence a visualizing agent can be applied to the gel, so that the separation can be viewed and analyzed. This technique can be used to check for the molecular weight of molecules or to check and confirm the amplification of a DNA fragment via PCR.

It can also be used as a preliminary technique to prep a sample that has to be further analyzed using various techniques such as RFLP, DNA sequencing, mass spectroscopy, blotting techniques, etc.

❖ Gel electrophoresis refers to the sorting of molecules, while moving through a matrix subjected to electromotive force. The gel used in this technique is made up of a cross-linked polymer, whose composition and porosity is based on the type and weight of the sample to be analyzed.

It shows presence of "wells" that are used to insert the sample. The solidified gel is placed within the electrophoretic unit, and the buffer is poured in till the gel is sufficiently submerged. The buffer acts as the source of cations and anions, and also as the medium for the ionic and electric current. It also maintains the pH of the gel.

Next, the sample is mixed with a specific quantity of dyes like bromophenol blue (BPB) and ethodium bromide (EtBr), and then inserted into the wells with the help of a micropipette. Each unique sample is loaded in a new well, and a ladder is also loaded in either the first or the last well, adjoining the samples.

❖ Adding the bromophenol blue to the sample provides density to the sample and also allows the migration of the sample to be visualized. The migration of the sample can be tracked by the migration of this dye in the gel. This is important, to know when the electrophoresis has been completed, as the samples are usually colorless.

Ethidium bromide is added only in case of DNA and RNA samples. It is an intercalating agent, that holds onto the nucleic acid molecules, and helps in visualizing the fragments under the action of UV light.

The ladder is a mixed solution containing various specific length fragments of the molecule to be analyzed, i.e if the sample is being analyzed for DNA, a DNA ladder is used, and in the case of a protein, a protein ladder is used.

The ladder helps in deciphering the molecular weight of the sample fragments, as the weight of each fragment of the ladder is already known.

❖ Once the samples and the ladder have been loaded, the unit is covered and attached to an electric current.

While attaching the electrodes, one must make sure that the wells are placed near the cathode, as the direction of an electric current is from negative to positive. If the wells are placed at the anode, the current will cause the molecules to migrate out of the gel towards the anode instead of migrating through the gel, towards the other end of the gel.

After the gel and unit have been satisfactorily set up, the electric current is switched on and kept at a constant voltage between 100 to 130 volts. During the course of the electrophoresis, if the gel heats up and starts melting due to the electric current, the voltage should be reduced.

The voltage applied to the gel-buffer system is indirectly proportional to the time taken for the completion of the run. The higher the voltage, the faster the run, and the lower the voltage, the slower the run.

❖ In a Electrophoretic run, the larger molecules migrate slower than the smaller molecules, hence the different sized fragments migrate at different speeds, causing a separation. Fragments of similar length will run parallel in individual lanes.

Attention should be paid to the migration of the dye in the gel, during the course of electrophoresis, and when the dye front approaches the end of the gel, the electric current should be stopped, and the gel can be taken out of the unit to be visualized.

In case of a gel with a nucleic acid sample, the gel can be viewed under UV light (action of EtBr), and in case of proteins, various stains like coomassie brilliant blue stain or silver staining can be used. If the fragments are to be used for further experiments, the specific area of the gel can be cut out and used to recover that fragment of bio-molecule.

Two types of gels are used for electrophoresis―polyacrylamide and agarose. Polyacrylamide gels are usually used for proteins, and they exhibit a very high resolving power for small fragments of DNA (5-500 bp).

Agarose gels have a lower resolving power for DNA but show a greater range of separation, and therefore are used in case of DNA fragments in the range of 50-20,000 basepairs (bp).

Polyacrylamide gels are run in a vertical configuration while agarose gels are run horizontally in a submarine mode. They also differ in their casting methodology, as agarose sets thermally, while polyacrylamide forms in a chemical polymerization reaction.

Agarose is obtained from a natural polysaccharide polymer extracted from red seaweed. Agarose is a gel composed of long unbranched chains of uncharged carbohydrates, and hence exhibits the presence of pores. These pores are utilized by the molecules to migrate.

However, the pore size is not uniform, but is optimal for the separation of proteins larger than 200 kDa. This gel can also be used for the separation of DNA fragments ranging from 50 base pair to several mega bases. The distance between the separated bands of different lengths is dictated by the percent agarose in the gel.

A higher percentage offers a clearer resolution but requires a longer run time. The percentage of agarose also depends on the size of the molecule. A larger molecule would require large pores to migrate through, and hence would require a lower percentage of agarose.

0.7% Agarose gels are good for the resolution of large 5-10kb DNA fragments, and 2% gels are good for resolving small 0.2-1kb fragments. These percentages of gels are achieved by digesting and dissolving the agarose in the electrophoresis buffer.

Very tiny fragments can be separated using 3% gels but a better resolution can be achieved using a polyacrylamide gel.

Polyacrylamide is a polymerized gel containing acrylamide, which is a neurotoxin and hence must be handled with caution. It is usually used in polyacrylamide gel electrophoresis (PAGE) for separating proteins of sizes 5 to 2,000 kDa.

This gel provides a uniform pore size due to its cross-linked nature, and the pore size can be manipulated by adjusting the concentrations of acrylamide and bis-acrylamide used while creating the gel. Proteins separated on this gel can be transferred onto a nitrocellulose or PVDF membrane for the purpose of carrying out western blotting.

The gel setup is made in 2 parts - stacking gel and resolving gel. These gels are layered on top of each other, with the stacking gel on top and the resolving gel below in a vertical manner.

These gels are made by the chemical cross linking of the acrylamide and bis-acrylamide due to the action of tetramethylethylenediamine (TEMED) and ammonium persulfate (APS). The stacking gel (5%) contains the wells in which the sample and ladder is loaded.

The stacking gel acts to stack the proteins together, so they may enter and migrate through the resolving gel uniformly.

The lower resolving gel has varying concentrations of 5%, 8%, 10%, 12% and 15%, depending on the size of the protein to be resolved.

The weight of the protein is inversely proportional to the percentage of the resolving gel. Higher percentage of gel is used with proteins of a small molecular weight, and vice versa. A higher and clearer resolution can be obtained by altering the buffer system appropriately.

These gels are viewed by silver staining or by staining with coomassie brilliant blue (CBB).

A non-toxic medium for protein electrophoresis can be found in the form of starch gels. These gels are more opaque than acrylamide or agarose, and can separate non-denatured proteins according to charge and size. The gel can be visualized by treatment with Naphthol Black or Amido Black staining. The commonly used percentages of starch gels are 5% to 10%.

Denaturing gels refer to gels that are run under conditions that denature the analyte, i.e. the natural structure of the molecule is disrupted, causing it to become linear. This allows analysis of only the primary structure of the molecules.

Denaturation of nucleic acids is done by including urea in the buffer solution, and proteins are denatured by using sodium dodecyl sulfate (SDS) in the buffer for PAGE. For a complete denaturation of proteins, beta-mercaptoethanol or dithiothreitol is added and the process is then called reducing PAGE.

Denaturing is essential in the case of RNA molecules, to determine the exact molecular weight. This is due to RNA's affinity towards forming bonds with DNA. Denaturing conditions prevent the formation of any such bonds, allowing the RNA to migrate freely. RNA is denatured via the use of urea, dimethyl sulfoxide (DMSO), and glyoxal.

Denaturing electrophoresis methods for protein include SDS-PAGE, and those for DNA and RNA include denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient electrophoresis (TTGE).

Native gels refer to gels that are run in conditions that preserve and maintain the analyte's natural structure. This allows the migration of the molecule through the gel to be affected by the physical size of the complex, allowing the analysis of all four levels of molecular structure.

Detergents used for the purpose of isolating the bio-molecules, are used in specific quantities so as to avoid any unwanted denaturation.

Due to the preservation of the natural native structure of the molecules, it becomes possible to separate them based on molecular mass, intrinsic charge, and also the cross-sectional area of the molecule. Proteins separated under these conditions must be visualized using enzyme-linked staining as well as conventional staining methods.

These conditions are maintained when one wishes to conduct proteomic and metallomic studies. They can also be used to scan genes (DNA) for unknown mutations and polymorphisms in Single-strand conformation polymorphism (SSCP) analysis.

Buffers provide ions for the conductance of the electric current, and also maintain a constant pH. The most common buffers used for nucleic acids are Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE). The borate in TBE buffer can polymerize, and interact with cis diols such as those found in RNA.

TAE has the lowest buffering capacity but provides the best resolution for larger DNA. Hence, TAE is mostly used instead of TBE. For proteins, a specific buffer is used depending on the type of PAGE being run. A denaturing PAGE is run using a SDS buffer, whereas, a native PAGE is run using a non-denaturing buffer.

Also, protein separations are done in a "discontinuous" system within the gel. In such a gel system, an ion gradient is formed initially, causing the proteins to focus into a single sharp band (isotachophoresis). This band is then separated according to size in the lower, "resolving" region of the gel.

▶ 2-D PAGE
▶ Native-PAGE
▶ Electroblotting
▶ Isotachophoresis
▶ Affinity electrophoresis
▶ Capillary electrophoresis
▶ Isoelectric focusing (IEF)
▶ Sequencing electrophoresis
▶ Pulsed-field gel electrophoresis
▶ Agarose gel electrophoresis (AGE)
▶ Electrophoretic mobility shift assay (EMSA)

▶ Gradient gel electrophoresis (DGGE)
▶ Single-strand conformation polymorphism (SSCP)
▶ SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)
▶ Temporal temperature gradient electrophoresis (TTGE)
▶ Restriction fragment length polymorphism (RFLP) analysis
▶ Quantitative preparative native continuous PAGE (QPNC-PAGE)

★ Electrophoresis of DNA fragments produced due to digestion by restriction enzymes, helps in estimating the size of DNA fragments. This data allows one to construct a restriction map of that particular DNA sample.

★ PCR products can be loaded onto agarose gels and electrophoresed to confirm the amplification of the DNA fragment, and to point out genetic anomalies or to identify the DNA pattern in case of genetic fingerprinting.

★ Proteins and DNA/RNA can be electrophoresed to separate the different fragments, and this can then be used to carry out Western and Southern/Northern Blotting techniques.

★ RFLP analysis can be carried out using gel electrophoresis. This analysis compares the banding patterns of the molecules between different individuals, and has numerous uses in the field of forensics, molecular biology, genetics, microbiology and biochemistry.

★ RNA samples are electrophoresed to check for DNA contamination and RNA degradation..

★ Protein samples are electrophoresed and subjected to ligand interaction assays to help characterize and identify different proteins in the mixed sample.

★ Electrophoresis is also used to determine the various binding constants and structural features of proteins.

From the technique's development, in the 1950s', till date, the technique's versatile nature has allowed it to undergo various modifications to give rise to a multitude of diverse methods and applications. This diversification is helped and enhanced by the increasing strides being made in the advancement of the understanding and application of the sciences.