Gel Electrophoresis Gel electrophoresis is a method that separates macromolecules, either nucleic...

Gel Gel ElectrophoresisElectrophoresis

Gel electrophoresis is a method Gel electrophoresis is a method that separates macromolecules, that separates macromolecules, either nucleic acids or protein.either nucleic acids or protein.

Electrophoresis describes the Electrophoresis describes the migration of charged particles migration of charged particles under the influence of an under the influence of an electric field.electric field.

A gel is a colloid in a solid form.A gel is a colloid in a solid form.Kris-itd.unair.ac.id (for education purpose only)

Gel electrophoresis refers to the Gel electrophoresis refers to the technique in which molecules are technique in which molecules are forced across a span of gel, forced across a span of gel, motivated by an electrical current.motivated by an electrical current.

Biological molecules exist in a Biological molecules exist in a solution as cations (+) or anions.solution as cations (+) or anions.

Gel electrophoresis is a technique Gel electrophoresis is a technique used for separation of nucleic acids used for separation of nucleic acids and proteins.and proteins.

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Separation of macro molecules Separation of macro molecules depend upon two forces; charge and depend upon two forces; charge and mass. During electrophoresis rate of mass. During electrophoresis rate of movement of macromolecules movement of macromolecules through the electric field depends on through the electric field depends on the strength of the field, size and the strength of the field, size and shape of the molecules, relative shape of the molecules, relative hydrophobicity of the sample and on hydrophobicity of the sample and on the ionic strength and temperature the ionic strength and temperature of the buffer in which the molecules of the buffer in which the molecules are moving. are moving.


Two Basic Types of Two Basic Types of Materials are used to make Materials are used to make

GelsGels AgaroseAgarose PolyacrylamidePolyacrylamide Agarose is a natural colloid Agarose is a natural colloid

extracted from sea weed.extracted from sea weed. Agarose gels can be processed Agarose gels can be processed

faster than polyacrylamide gels.faster than polyacrylamide gels.


Agarose is a linear Agarose is a linear polysaccharide made up of the polysaccharide made up of the basic repeat unit agrobiose, basic repeat unit agrobiose, which comprises alternating which comprises alternating units of galactose and 3, 6 units of galactose and 3, 6 anhydrogalactose.anhydrogalactose.

Structure of Agarose GelStructure of Agarose Gel


Apparatus Used In Gel Apparatus Used In Gel Electrophoresis.Electrophoresis.


PAGE, technique was introduced by PAGE, technique was introduced by Raymond and Weintraub (1959).Raymond and Weintraub (1959).

Polyacrylamide is the same Polyacrylamide is the same material that is used for skin material that is used for skin electrodes and in soft contact lense.electrodes and in soft contact lense.

By controlling the percentage (from By controlling the percentage (from 3% to 30%) precise pore sizes can 3% to 30%) precise pore sizes can be obtained, usually from 5 to 2000 be obtained, usually from 5 to 2000 kdal.kdal.

PolyacrylamidePolyacrylamide


Gradient gels provide Gradient gels provide continuous decrease in pore size continuous decrease in pore size from the top to the bottom of the from the top to the bottom of the gel, resulting in thin bands.gel, resulting in thin bands.

Polyacrylamide gels offer Polyacrylamide gels offer greater, flexibility and move greater, flexibility and move sharply defined banding than sharply defined banding than agarose gels.agarose gels.


Polyacrylamide Gel Polyacrylamide Gel Electrophoresis System Electrophoresis System

PAGE SystemPAGE System Different Samples are loaded in wells Different Samples are loaded in wells

at the top of the polyacrylamide Gel.at the top of the polyacrylamide Gel. Proteins move into the Gel when an Proteins move into the Gel when an

electric field is applied. electric field is applied. Proteins can be visualized after Proteins can be visualized after

electrophoresis by treating the Gel electrophoresis by treating the Gel with the stain such as Coomassie with the stain such as Coomassie blue.blue.


Nucleic AcidsNucleic Acids

Nucleic acids transmit hereditary Nucleic acids transmit hereditary information and determine which proteins a information and determine which proteins a cell manufactures.cell manufactures.

DNA comprises the genes.DNA comprises the genes. RNA functions in the process of protein RNA functions in the process of protein

synthesis.synthesis. Nucleic acids are large, complex molecules Nucleic acids are large, complex molecules

were first isolated by Miescher in 1870.were first isolated by Miescher in 1870.


Electrophoresis of Electrophoresis of Nucleic AcidsNucleic Acids

Larger pieces of DNA collide Larger pieces of DNA collide with the gel matrix more often with the gel matrix more often are slowed down, while smaller are slowed down, while smaller pieces of DNA move through pieces of DNA move through more quickly.more quickly.

By using gel electrophoreses, By using gel electrophoreses, biologists can tell which gene biologists can tell which gene is based upon the sizes of the is based upon the sizes of the fragments generated when a fragments generated when a gene is treated with restriction gene is treated with restriction enzyme.enzyme.Kris-itd.unair.ac.id (for education purpose

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DNA BandsDNA Bands


ProteinsProteins Proteins are of central importance Proteins are of central importance

in the structure and function of all in the structure and function of all living organisms.living organisms.

Electrophoresis of Electrophoresis of ProteinsProteins

Methods for separating Methods for separating proteins take advantage of proteins take advantage of properties such as charge, size properties such as charge, size and solubility.and solubility.

Proteins can also be separated Proteins can also be separated on the basis of their binding on the basis of their binding properties.properties.


The source of protein is The source of protein is generally tissue or microbial generally tissue or microbial cells.cells.

Variety of methods is available Variety of methods is available for separation of proteins, such for separation of proteins, such as Ion exchange as Ion exchange chromatography other chromatography other chromatographic methods take chromatographic methods take advantage of differences in advantage of differences in size, binding, affinity and size, binding, affinity and solubility.solubility.Kris-itd.unair.ac.id (for education purpose

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Another method is available for Another method is available for separation of proteins, a separation of proteins, a process called electrophoresis.process called electrophoresis.

Gel electrophoresis is Gel electrophoresis is advantageous, because in this advantageous, because in this process proteins can be process proteins can be visualized as well as separated.visualized as well as separated.

The net charge of a protein will The net charge of a protein will depend on its amino acid depend on its amino acid composition.composition.


Protein even with a variation of Protein even with a variation of one amino acids will have a one amino acids will have a different overall charge, and different overall charge, and thus are electrophoretically thus are electrophoretically distinguishable.distinguishable.

The gel results will show that The gel results will show that some of the high molecular some of the high molecular weight bands from the sample weight bands from the sample not treated with the disulfide not treated with the disulfide reducing agent are missing in reducing agent are missing in the sample treated with the the sample treated with the disulfide reducing agent.disulfide reducing agent.


The break up of complex The break up of complex proteins into their respective proteins into their respective polypeptides allows us to study polypeptides allows us to study the structure of proteins that the structure of proteins that result from the interaction of result from the interaction of several genes.several genes.

A gene is a discrete unit of A gene is a discrete unit of hereditary information that hereditary information that usually specifies a protein.usually specifies a protein.


A single gene provides the A single gene provides the genetic code for only one genetic code for only one polypeptide.polypeptide.

A molecular weight protein A molecular weight protein marker is used to prepare a marker is used to prepare a standard separation cure with standard separation cure with which various unknown which various unknown proteins or polypeptide proteins or polypeptide fractions can be identified.fractions can be identified.Kris-itd.unair.ac.id (for education purpose

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ApplicationsApplicationsDNA Fingerprinting on DNA Fingerprinting on

TrialTrial An individuals DNA is as An individuals DNA is as

distinctive as a fingerprint.distinctive as a fingerprint. DNA samples can be obtained DNA samples can be obtained

from the trace amounts of from the trace amounts of blood or semen.blood or semen.

DNA samples can be separated DNA samples can be separated by using gel electrophoresis.by using gel electrophoresis.


In mid-1980s, genetic In mid-1980s, genetic fingerprinting has rapidly fingerprinting has rapidly become a widely used court become a widely used court room tool.room tool.

In 1988 the first person in the In 1988 the first person in the USA was executed based on USA was executed based on DNA technology. DNA technology.


The Human Genome The Human Genome ProjectProject

Four complementary Four complementary approaches are being usedapproaches are being used

1.1. Genetic mapping of the Genetic mapping of the human genome. human genome.

2.2. Physical mapping of the Physical mapping of the human genome.human genome.

3.3. Sequencing the human Sequencing the human genome.genome.

4.4. Analyzing the genomes of Analyzing the genomes of other species.other species.


DNA TechnologyDNA Technology

We are now using We are now using biotechnology to study the biotechnology to study the basic processes of life, basic processes of life, diagnose illnesses, and develop diagnose illnesses, and develop new treatments for diseases.new treatments for diseases.

Some of the tools of Some of the tools of biotechnology are natural biotechnology are natural components of cells.components of cells.

Restriction enzymes are made Restriction enzymes are made by bacteria to protect by bacteria to protect themselves from viruses. They themselves from viruses. They inactive the viral DNA by inactive the viral DNA by cutting it in specific places.cutting it in specific places.


DNA ligase is an enzyme that DNA ligase is an enzyme that exist in all cells.exist in all cells.

Restriction enzymes can be Restriction enzymes can be used to cut DNA at specific used to cut DNA at specific sequences called recognition sequences called recognition sites.sites.

Recombinant DNA sequences Recombinant DNA sequences contain genes form two or contain genes form two or more organisms. more organisms.


By using DNA technology By using DNA technology researchers have gained the researchers have gained the ability to diagnose diseases ability to diagnose diseases such as sickle cell anemia, such as sickle cell anemia, Hutington’s chorea early in the Hutington’s chorea early in the course of the disease.course of the disease.

The most important The most important achievements resulting form achievements resulting form recombinant DNA technology recombinant DNA technology have been advances in our have been advances in our basic understanding of basic understanding of eukaryotic molecular biology.eukaryotic molecular biology.


DNA technology is in the DNA technology is in the process of revolutionizing process of revolutionizing biological research, human biological research, human medicine, criminal law and medicine, criminal law and agriculture.agriculture.


Uses of Gel Uses of Gel Electrophoresis Electrophoresis

Identification of particular Identification of particular DNA molecules by the band DNA molecules by the band patterns.patterns.

Viral DNA, plasmid DNA and Viral DNA, plasmid DNA and particular segments of particular segments of chromosomal DNA can all be chromosomal DNA can all be identified in this way.identified in this way.

Isolation and purification of Isolation and purification of individual fragments individual fragments containing interesting genes.containing interesting genes.

It determines the genetic It determines the genetic differences and evolutionary differences and evolutionary relationship among species of relationship among species of plants and animals.plants and animals.


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Gel Electrophoresis Gel electrophoresis is a method that separates macromolecules, either nucleic...

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