GEL ELECTROPHORESIS

WHAT IS GEL ELECTROPHORESIS ?

Gel electrophoresis is a method that separates macromoleculeseither nucleic acids or proteins-on the basis of size, electric charge, and other physical properties. The term electrophoresis describes the migration of charged particle under the influence of an electric field.

Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionisable groups and, therefore, at any given pH, exist in solution as electically charged species either as cations (+) or anions (-). Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode.

HOW DOES THIS TECHNIQUE WORK?

Separation of large (macro) molecules depends upon two forces: charge and mass. When a biological sample, such as proteins or DNA, is mixed in a buffer solution and applied to a gel, these two forces act together. The electrical current from one electrode repels the molecules while the other electrode simultaneously attracts the molecules. The frictional force of the gel material acts as a "molecular sieve," separating the molecules by size.Their rate of migration through the electric field depends on the strength of the field, size and shape of the molecules, relative hydrophobicity of the samples, and on the ionic strength and temperature of the buffer in which the molecules are moving. After staining, the separated macromolecules in each lane can be seen in a series of bands spread from one end of the gel to the other.

APPLICATIONS
Gel Electrophoresis is one of the staple tools in molecular biology and is of critical value in many aspects of genetic manipulation and study. One use is the identification of particular DNA molecules by the band patterns they yield in gel electrophoresis after being cut with various restriction enzymes. Viral DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this way. Another use is the isolation and purification of individual fragments containing interesting genes, which can be recovered from the gel with full biological activity.

 to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid. The protein molecules in a sample of fish muscle tissue and plant grain endosperm tissue can be separated according to their individual molecular mass and compared to samples that have been treated with a reducing agent, such as 2-mercaptoethanol. Complex proteins (composed of two or more polypeptide chains) can be broken down into their respective polypeptide fractions. A reducing agent breaks the disulfide bonds that hold the polypeptide together.

Electrophoresis describes the migration of a charged particle under the influence of an electric field. Under the influence of an electric field, the charged particles ( aminoacids, peptides, proteins, nucleic acids) will migrate to the cathode or anode depending upon the net charge Equipment of electrophoesis -----Power pack Electrophoresis units

Power pack supplies direct current between the electrodes. Electrophoresis unit consists of gel formed between 2 glass plates clamped together, but held apart by plastic spacers. Gel dimensions are 12 cm and 14 cm with a thickness of 1-2 mm. A Plastic comb is placed in the gel sol and removed after polymerisation to provide loading wells for samples.

 Electrophoresis is carried out in an appropriate buffer- maintains a constant state of ionization of the molecules being separated. Th lower electrophoresis tank buffer surrounds the gel plate and affords some cooling of gel plates.

AGAROSE GELS
It is a linear polysacharide of molecular mass about 12000, made of alternating units of galactose and 3, 6- anhydrogalactose. The gelling properties attributed to inter and intramolecular hydrogen bonding within and between long agarose chains. The pore size of the gel controlled by the initial concentration of agarose. Low conc of agarose -- Large pore size High conc of agarose -- Small pore size Used for the electrophoresis of proteins and nucleic acids. Due to the low M. P of Agarose, (62-65OC ) the gels can be reliquified by heating to 65oC and so DNA samples separated in the gel can be returned to solution and recovered.

Electrophoresis of Nucleic Acids

An agarose or polyacrylamide gel is loaded with the DNA fragments and current is passed through the gel. Since DNA is negatively charged, it will migrate towards the positive pole. The DNA will not migrate at the same rate, however. Larger pieces of DNA collide with the gel matrix more often and are slowed down, while smaller pieces of DNA move through more quickly. Since different genes have different nucleotide sequences, restriction enzymes will cut them at different places, generating different size DNA fragments.

By using gel electrorphoresis, biologists can tell which gene is which based upon the sizes of the fragments generated when a gene is treated with a restriction enzyme.

AGAROSE GEL ELECTROPHORESIS OF DNA

Most DNA molecules and their fragments are larger than proteins. Most DNA fragments are unable to enter a polyacrylamide gel, hence the larger pore size of agarose gel is required. Since the charge per unit length of DNA in any given fragment of DNA is the same ( due to the phosphate groups ) all DNA samples should move towards the anode with the same mobility under an applied electric field. The mobility of a DNA fragment depends on the size, the smallest molecules moving fast. Gel conc should be chosen to suit the size range of molecules to be separated.

0.3 % gels Separates DNA molecules between 5 and 60 Kb size. 2 % gels -- Separates DNA molecules between 0.1 and 3 kb size. 0.8 % gels Separates in the range of 0.5- 10 kb size. The M r of a DNA fragment can be determined by running a number of standard markers of known Mol.mass on the same gel. Bromophenol Blue is used as the tracking dye in the sample solvent. No stacking gel is needed.

For viewing the gel , the fluroscent dye ethidium Bromide is used and then viewed under UV light. EB is a cyclic planar mol that binds between the stacked base pairs and fluoresce under UV light.

POLYACRYLAMIDE GELS
Electrophoresis in acrylamide gels - PAGE Cross linked polyacrylamide gels formed from polymerisation of acrylamide monomer in the presence of smaller amounts of N, Nmethylene Bis acrylamide. 2 acrylamide molecules are linked by a methylene group and used as a cross linking agent. Acrylamide monomers is polymerized in a head to tail fashion into long chains and occasionally a Bis acrylamide molecule is built into the growing chain, thus introducing a second site for chain extension. APS -- Polymerization of acrylamide. TEMED Speeds up the polymerization process.

Pore size of the gel determined by the concentration of acrylamide and polyacrylamide.
Low percent gels ( eg 3 % ) with large pore size used in the electrophoresis of proteins.Also used for the electrophoresis of DNA for sequencing.

ELECTROPHORESIS OF PROTEINS
Amino acids differ not only in R-group characteristics but also in molecular weight. Different amino acids are linked together in a linear chain by peptide bonds in various combinations and sequences to form specific proteins. A protien may be comprised of amino acids from all of the categories. The net charge of a protein will depend on its amino acid composition. If it has more positively charged amino acids such that the sum of the positive charges exceeds the sum of the negative charges, the protein will have an overall positive charge and migrate to the cathode (negatively charged electrode) in an electrical field. Proteins even with a variation of one amino acids will have a different overall charge, and thus are electrophoretically distinguishable.

 Proteins can be visualized as well as separated, permitting a researcher to estimate quickly the number of proteins in a mixture or the degree of purity of a particular protein preparation. Also, gel electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. The break up of complex proteins into their respective polypeptides allows us to study the structure of proteins that result from the interaction of several genes. A gene is a discrete unit of hereditary information that usually specifies a protein. A single gene provides the genetic code for only one polypeptide. Thus, a protein consisting of four polypeptides requires the interaction of four genes to synthesize that specific protein. A molecular weight protein marker is used to prepare a standard separation curve with which various unknown proteins or polypeptide fractions can be identified.

PAGE OF PROTEINS
Analyse protein mixtures. Based on the separation of proteins according to size. Used to determine the mol mass of proteins. SDS is an anionic detergent. Samples first boiled for 5 min in sample buffer containing beta mercaptoethanol and SDS. Mercaptoethanol reduces disulfide bridges present that are holding together the tertiary structure of proteins SDS denatures the proteins. Each protein is fully denatured and opens up into a rod like structure with a series of vely charged SDS molecules along the peptide chain. Bromophenol blue Tracking Dye Glycerol Gives density allowing the sample to settle through the buffer to the bottom when injected into the loading well.

2 types of gel, Stacking and resolving. Purpose of stacking gel Concentrate the protein into a sharp band before it enters the separating gel. Achieved by Isotachaphoresis. As the protein moves towards the anode, The smaller the protein, the more easily it can pass through the pores of the gel whereas the larger proteins are retarded by the frictional resistance by the sieving efffect of the gel. Stained by CBB. Destained overnight and fixed in fixative.

Choice of the gel Depends on the size of the protein being studied. Separating gel 15% Polyacrylamide For separation of proteins of Mol mass of 10,000 1,00,000 7.5 gel 0r 10% gel For separation of protein of mol mass 1,50,000 Std proteins of known Mr run on the same gel. By plotting a graph of distance moved against log Mr for each std protein, a calibration curve constructed. Distance moved by the protein of unknown Mr measured, log Mr calculated and Mr determined from the caliberation curve.

SIGNIFICANCE

To assess the purity of the sample. A pure protein 1 single band 2 subunits 2 bands Mol mass of the protein

NATIVE GEL
To detect a particular protein on the basis of its biological activity. So denaturing conditions cannot be uesd. SDS is absent and proteins are not denatured. Since all the proteins in the sample carry their native charge, proteins separate acording to their different electrophoretic mobilities and the sieving effect of the gel. The enzyme of interest can be identified by incubating the gel in an appropriate substrate solution such that a coloured product is produced at the site of the enzyme.

GRADIENT GELS
The acrylamide gel forms a gradient varying in conc from 5% at the top of the gel to 25% acrylamide at the bottom of the gel. The higher conc of acrylamide is poured between the glass plates first and a continuous gradient of decreasing conc of acrylamide follows. At the top of the gel, there is a larger pore size and at the bottom the pore size decreases. Advantages A much greater range of protein Mr values can be separated than on a fixed percent gel. Proteins with similar Mr values can be resolved.

ISO ELECTRIC FOCUSING GEL ( IEF ) GEL

For separation of amphoteric substances like proteins based on the different isoelectric points. Utilizes horizontal gels on glass plates or plastic sheets. P. D is applied across the gel that contains a PH gradient. The PH gradient is formed by the introduction of ampholytes which are complex mixtures of synthetic polyamino poly carboxylic acids. Ampholytes come in a wide range, ( wide band ( PH- 3- 10 ) or narrow bands ( PH- 7-8 ) ) PH range is chosen such that the sample being separated will have their isoelectric points within this range.

The pI of a particular protein determined by running a mixture of proteins of known pI on the same gel. After staining, the distance of each band from one electrode is measured and a graph of distance for each protein againt its pI plotted. The pI of the unknown protein can be determined from the position on the gel. Particularly useful in separating isoenzymes.For studying heterogenesity of a protein, like small charge introduced on each mol can be checked by IEF.

TWO DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS

Combines the technique of IEF and size separation by SDS polyacrylamide gel electrophoresis.

When combined, it is the most sophisticated analytical method for separating proteins.this method is capable of resolving 500010000 spots of proteins in a cell.

DETECTION, ESTIMATION AND RECOVERY OF PROTEINS FROM GELS

Dye used is CBB R- 250 Usually carried out in 0.1 % CBB in methanol: water: glacial acetic acid. Acid methanol mixture acts as a denaturant to precipitate or fix the protein onto the gel which prevents the protein from being washed away while being stained. It is highly sensitive, a very weakly staining band on the polyacrylamide gel will corespond to 100 ng of protein.

SILVER STAINING
Silver ions are reduced to metallic silver on the protein, where the silver is deposited to give a black band. It can be used immediately after electrophoresis or after staining with CBB. The major bands in theis case can be visualised by CBB and the minor bands by silver staining. The silver stain is 100 times more sensitive than CBB detecting proteins upto 1 ng amounts.

LECTIN STAINING
`They are protein molecules that bind different types of carbohydrates They recognize mannose, fucose or terminal glucosamine of the carbohydrate side chains of glycoproteins. Coloured bands appear at the point where the lectins bind if each track is incubated with a different lectin, washed, incubated with horseradish peroxidase linked antibody to lectin and then peroxidase substrate added. The type of glycosylation of the glycoprotein can be determined as above.

DNA SEQUENCING GELS

The final analysis involves separating ss DNA molecules shorter than 1000nt and differing by 1 nt. 3.5% polyacrylamide gels are used to separate DNA in the range of 80-1000 nt. 12% gels used to resolve 20-100 nt. Sequencing gels run in the presence of denaturing agents like urea, formamide etc. The DNA bands of interest can be cut from the gel and DNA recovered by electroelution,DNA finally recovered by pptating the supernatent with ethanol.

ELECTROPHORESIS OF RNA
gel electrophoresis 1 hour For checking the integrity of RNA immediately after extraction. 18s rRNA and 28s rRNA can be resolved easily. DNA contamination Carried out in agarose gels based on size. 2% agarose can also be seen easily. If the study objective is to determine the size of RNA then full denaturation of RNA is needed. Urea, formaldehyde, glyoxal etc are used. Sample is heated in the presence of denaturant prior to electrohporesis. After this, these agents form adducts with amino groups of guanine and uracil, prevents hydrogen bonding reformation during electrophoresis. Acrydine orange is uesd for staining. Also radiolabelled RNA can be used and detected by autoradiography.

PULSE FIELD GEL ELECTROPHORESIS

DNA fragments upto 2000 Kb can be separated. Allows rhe separation of whole chromosomes by separation. Involves electrophoresis of agarose where 2 electric fields are applied alternately at different angles for definite time periods. Activation of the first electric field causes coiled molecules to be stretched in the horizontal plane and start to move through the gel. Interruption of the first field and the application of the second field force the molecule to move in the new direction. Since there is length dependent relaxation behaviour, when a long chain mol undergoes conformational change in an electric field, the smaller a mol, the quicker it realligns itself with the new field and continue to move through the gel.

Larger mol take longer time to reallign. In this way, with continuous reversing of the field, smaller mols draw ahead of larger mols and separate accoridng to their size.

THANK YOU