The document discusses electrophoresis, which is the movement of charged particles in an electric field. It migrates towards the oppositely charged electrode. Electrophoresis is used to separate biological molecules like proteins. It explains the principles and types of electrophoresis like paper, gel, and isolectric focusing electrophoresis. It provides details about agarose gel electrophoresis including properties, buffers used, procedure, and applications. It also briefly discusses polyacrylamide gel electrophoresis.
Electrophoresis-PAPER ELECTROPHORESIS,GEL ELCTROPHORESIS, PAGE-SDS AND NON-SDSAmrutha Hari
The document discusses different electrophoresis techniques including paper electrophoresis, gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and non-SDS PAGE. It describes the basic principles, apparatus, procedures, applications and advantages/disadvantages of each technique. Paper electrophoresis is a simple method that uses filter paper as the support medium while gel electrophoresis uses gels like agarose or polyacrylamide as the support medium. SDS-PAGE and PAGE are variations that separate molecules based on their molecular weight or size.
This document discusses different types of electrophoresis techniques. It begins with an introduction to electrophoresis and its history. It then describes various electrophoresis techniques including gel electrophoresis using agarose gel or polyacrylamide gel, capillary electrophoresis, isoelectric focusing, two-dimensional electrophoresis, and immuno electrophoresis. The document explains the basic principles of electrophoresis and how it uses an applied electric field to separate biomolecules like proteins, nucleic acids, and carbohydrates based on their charge and size. It concludes with some applications of electrophoresis techniques.
1. Electrophoresis is a technique used to separate charged molecules like proteins and nucleic acids by using an electric field to move them through a medium like gel or paper.
2. The rate at which molecules move depends on factors like their charge, size, and shape. Larger or more irregularly shaped molecules move more slowly.
3. Common types of electrophoresis include paper electrophoresis, cellulose acetate electrophoresis, and gel electrophoresis using agarose or polyacrylamide gels. Gel electrophoresis is often used to separate proteins and DNA fragments by size.
This document provides an overview of electrophoresis techniques. It begins with an introduction to electrophoresis as the migration of charged particles under an electric field. It then discusses various factors that affect electrophoresis like net charge, size, strength of electric field, buffer properties, and temperature. It describes different types of electrophoresis like paper, gel, cellulose acetate, isoelectric focusing, and moving boundary electrophoresis. Specific details are given about gel electrophoresis techniques like agarose, polyacrylamide gel, and SDS-PAGE electrophoresis. Other techniques like isolectric focusing, two-dimensional electrophoresis, isotachophoresis, pulsed field gel electrophoresis, and immunoelectrophoresis are also summarized briefly in 3 sentences
Electrophoresis is a technique used to separate charged molecules like proteins and DNA. It works by applying an electric current which causes the molecules to migrate through a buffer or gel at different rates depending on their size and charge. The document discusses the principles of electrophoresis, different types of electrophoresis like agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE), and factors that influence molecule migration like pH, molecular weight, and net charge.
Electrophoresis is the migration of charged particles through an electric field towards the electrode of opposite charge. It works by separating molecules based on their size and charge. Key factors that affect electrophoresis include the net charge and size/shape of molecules, as well as the ionic strength, pH, temperature, and type of support medium used. Common applications include separating proteins, nucleic acids, and other biological molecules to analyze samples.
Poly acrylamide gel electrophoresis (page)Dharmesh Gol
This document provides an overview of polyacrylamide gel electrophoresis (PAGE). It describes how PAGE uses an electric field to separate charged biomolecules like proteins and nucleic acids based on their size and charge. The document discusses the principles and instrumentation of electrophoresis. It explains how polyacrylamide gels are made and the procedures for PAGE. Different types of PAGE like SDS-PAGE and native PAGE are also summarized.
Gel electrophoresis separates molecules like DNA, RNA, and proteins based on their size and charge. The molecules are placed in a gel matrix and an electric current is applied, causing them to migrate through the gel at different rates depending on their size and charge. Proteins require denaturing agents like SDS to give them a uniform negative charge and allow separation based primarily on size. Staining the gel after electrophoresis allows visualization of the separated molecules as distinct bands.
Electrophoresis-PAPER ELECTROPHORESIS,GEL ELCTROPHORESIS, PAGE-SDS AND NON-SDSAmrutha Hari
The document discusses different electrophoresis techniques including paper electrophoresis, gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and non-SDS PAGE. It describes the basic principles, apparatus, procedures, applications and advantages/disadvantages of each technique. Paper electrophoresis is a simple method that uses filter paper as the support medium while gel electrophoresis uses gels like agarose or polyacrylamide as the support medium. SDS-PAGE and PAGE are variations that separate molecules based on their molecular weight or size.
This document discusses different types of electrophoresis techniques. It begins with an introduction to electrophoresis and its history. It then describes various electrophoresis techniques including gel electrophoresis using agarose gel or polyacrylamide gel, capillary electrophoresis, isoelectric focusing, two-dimensional electrophoresis, and immuno electrophoresis. The document explains the basic principles of electrophoresis and how it uses an applied electric field to separate biomolecules like proteins, nucleic acids, and carbohydrates based on their charge and size. It concludes with some applications of electrophoresis techniques.
1. Electrophoresis is a technique used to separate charged molecules like proteins and nucleic acids by using an electric field to move them through a medium like gel or paper.
2. The rate at which molecules move depends on factors like their charge, size, and shape. Larger or more irregularly shaped molecules move more slowly.
3. Common types of electrophoresis include paper electrophoresis, cellulose acetate electrophoresis, and gel electrophoresis using agarose or polyacrylamide gels. Gel electrophoresis is often used to separate proteins and DNA fragments by size.
This document provides an overview of electrophoresis techniques. It begins with an introduction to electrophoresis as the migration of charged particles under an electric field. It then discusses various factors that affect electrophoresis like net charge, size, strength of electric field, buffer properties, and temperature. It describes different types of electrophoresis like paper, gel, cellulose acetate, isoelectric focusing, and moving boundary electrophoresis. Specific details are given about gel electrophoresis techniques like agarose, polyacrylamide gel, and SDS-PAGE electrophoresis. Other techniques like isolectric focusing, two-dimensional electrophoresis, isotachophoresis, pulsed field gel electrophoresis, and immunoelectrophoresis are also summarized briefly in 3 sentences
Electrophoresis is a technique used to separate charged molecules like proteins and DNA. It works by applying an electric current which causes the molecules to migrate through a buffer or gel at different rates depending on their size and charge. The document discusses the principles of electrophoresis, different types of electrophoresis like agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE), and factors that influence molecule migration like pH, molecular weight, and net charge.
Electrophoresis is the migration of charged particles through an electric field towards the electrode of opposite charge. It works by separating molecules based on their size and charge. Key factors that affect electrophoresis include the net charge and size/shape of molecules, as well as the ionic strength, pH, temperature, and type of support medium used. Common applications include separating proteins, nucleic acids, and other biological molecules to analyze samples.
Poly acrylamide gel electrophoresis (page)Dharmesh Gol
This document provides an overview of polyacrylamide gel electrophoresis (PAGE). It describes how PAGE uses an electric field to separate charged biomolecules like proteins and nucleic acids based on their size and charge. The document discusses the principles and instrumentation of electrophoresis. It explains how polyacrylamide gels are made and the procedures for PAGE. Different types of PAGE like SDS-PAGE and native PAGE are also summarized.
Gel electrophoresis separates molecules like DNA, RNA, and proteins based on their size and charge. The molecules are placed in a gel matrix and an electric current is applied, causing them to migrate through the gel at different rates depending on their size and charge. Proteins require denaturing agents like SDS to give them a uniform negative charge and allow separation based primarily on size. Staining the gel after electrophoresis allows visualization of the separated molecules as distinct bands.
Electrophoresis is a technique used to separate charged particles like proteins by using an electric field to migrate them through a medium like filter paper or gel based on factors like their size and charge. It can be used to separate plasma proteins, lipoproteins, and immunoglobulins into distinct bands. The document discusses several electrophoresis techniques including paper, gel, SDS-PAGE, isoelectric focusing, and immunoelectrophoresis and how they are used to separate biomolecules.
it is a short ppt. on Electrophoresis. Which gives it's defination, Types of electrophoresis ; Like:- Zone electrophoresis, Isoelectric focussing, Immunoelectrophoresis. and their images.
Electrophoresis is a technique used to separate charged particles such as proteins, nucleic acids, and other macromolecules. It works by applying an electric field to move these particles through a medium such as a gel or paper based on their size and charge. There are different types of electrophoresis including gel electrophoresis, paper electrophoresis, capillary electrophoresis, and isoelectric focusing which separates particles based on their isoelectric point. Electrophoresis has many applications in fields like forensics, molecular biology, genetics, and biochemistry to analyze proteins, DNA, RNA, and other biomolecules.
Electrophoresis is a technique used to separate charged molecules such as proteins and nucleic acids by using an electric field. There are different types of electrophoresis including zone electrophoresis using paper, cellulose acetate, or gel matrices, and moving boundary electrophoresis without a supporting medium. Gel electrophoresis techniques like agarose gel electrophoresis and SDS-PAGE are commonly used. Electrophoresis finds applications in fields like clinical testing, forensic analysis, and environmental monitoring by allowing separation and characterization of biomolecules.
Electrophoresis is a technique used to separate charged molecules like proteins and nucleic acids. It works by applying an electric field to move molecules through a buffer solution or gel based on their size and charge. There are several types of electrophoresis that use different supporting media like agarose gel, polyacrylamide gel, cellulose acetate, or paper to separate molecules. Factors like pH, buffer composition, strength of electric field, and temperature influence how molecules separate during electrophoresis. It has various applications in biomedical research and clinical diagnostics.
Modern pharmaceutical analytical technique ( Electrophoresis)KhushbooKunkulol
This document provides an overview of electrophoresis, including definitions, principles, types, and applications. It discusses four main types of electrophoresis: paper electrophoresis, gel electrophoresis, capillary electrophoresis, and iso-electric electrophoresis. Paper and gel electrophoresis are described as zone electrophoresis techniques that separate components into bands on a supporting medium like paper or gel. Capillary electrophoresis allows separation in free solution within a thin capillary tube. Iso-electric electrophoresis separates molecules based on their iso-electric point, the pH where they have no net charge. The document outlines the principles, instrumentation, and applications of each electrophoresis technique.
Electrophoresis is a method used to separate charged particles such as proteins and nucleic acids based on differences in their migration speed in an electric field. There are several types of electrophoresis including agarose gel electrophoresis, SDS-PAGE, isoelectric focusing, capillary electrophoresis, and two-dimensional electrophoresis. During electrophoresis, charged particles migrate through a gel or capillary towards an electrode with the rate of migration depending on factors like the particle's charge, size, and shape.
This document provides information about electrophoresis, including its theory, principle, types, and applications. It begins with an introduction that defines electrophoresis as the migration of charged particles in an electric field. It then describes the theory behind electrophoresis, explaining how an electrostatic force is balanced by a drag force to produce particle velocity. The main types of electrophoresis are described as slab electrophoresis, capillary electrophoresis, paper electrophoresis, and gel electrophoresis. Key applications are identified as protein analysis, DNA analysis, and vaccine analysis. The document concludes by emphasizing the importance of electrophoresis for research and forensic investigations.
Electrophoresis along with its history, application and types are discussed here to give a brief yet understanding outlook to the topic explained which is an important topic for the biotechnology as well as all biological research field.
Electrophoresis is a technique used to separate charged molecules like proteins and nucleic acids based on their size and charge. It involves applying an electric field to move molecules through a medium like agarose gel or capillary. Shorter/less charged molecules move faster and are separated from longer/more charged molecules. It is used in laboratories and clinical settings to analyze biological samples and diagnose conditions.
Gel electrophoresis is a method used to separate molecules like proteins and nucleic acids based on their physical properties. It works by applying an electric field to move charged molecules through a gel medium. Key aspects include applying samples to the gel, running electrophoresis with an electric current, and visualizing separated molecules by staining and quantification using a densitometer. Gel electrophoresis has applications in forensics, genetics, and biochemistry to analyze DNA, diagnose diseases, and study proteins. It separates biomolecules based on factors like size, shape, and charge.
This document provides information about electrophoresis. It begins by defining electrophoresis as the migration of charged particles or molecules under the influence of an electric field. Some purposes of electrophoresis are to determine components in a sample, separate them, and obtain information about electrical double layers or determine molecular weights. The principle described is that charged particles migrate in an electric field at a velocity depending on factors like charge, size, shape and applied current. Different types of electrophoresis are described like paper, gel, and isolectric focusing electrophoresis. Key aspects of each technique including apparatus, sample application, detection, and preparation of gels are explained. Two-dimensional electrophoresis combining isolectric focusing and SDS-PAGE is also summarized.
This document discusses electrophoresis, which is the movement of charged particles in an electric field. It separates molecules based on their charge and size. Key factors that affect migration rate are listed. The main requirements for electrophoresis are an electrophoresis tank, electrodes, power supply, buffer, and specimens like serum or plasma. Common electrophoresis techniques described include zone electrophoresis using paper or gel, isoelectric focusing, immuno electrophoresis, and SDS-PAGE which separates based on size. Clinical applications involve using electrophoresis to analyze conditions like liver disease or infections.
electrophoresis-
principle
types
details on paper electrophoresis
cellulose acetate electrophoresis
zone electrophoresis
SDS-PAGE
iso-electric focussing gel electrophoresis
two-dimensional gel electrophoresis
pulsed gel electrophoresis
isotachophoresis
capillary electrophoresis
microchip electrophoresis
This document provides an overview of electrophoresis techniques. It discusses the basic principles of electrophoresis, including that the rate of migration depends on the charge-to-mass ratio of particles. It describes various electrophoretic techniques such as moving boundary electrophoresis, zone electrophoresis using free solution, gel, or paper methods, as well as isoelectric focusing, isotachophoresis, and capillary electrophoresis. It provides details on how each technique is performed and discusses their applications and advantages.
Electrophoresis is a separation technique that is based on the movement of charged particles in an electric field.
Electrophoresis is an analytical method of separating charged particles based on their relative mobilities in an electric field
Microchip Electrophoresis is the new talk of the town, which revolutionize the field of electrophoresis. It is shown to be an attractive tool for time & cost saving development of a separation method for complex sample mixtures. It made possible the simultaneous separation of catecholamines and their cationic metabolites.
Gel electrophoresis is a method to separate biomolecules like proteins, nucleic acids, and lipids based on their charge and size. During gel electrophoresis, an electric current is applied across a gel, causing negatively charged molecules to migrate toward the positive electrode and positively charged molecules to migrate toward the negative electrode. Smaller molecules migrate faster through the gel than larger molecules. Factors like the charge, size, and shape of molecules, as well as the electric current, gel composition, and buffer used, determine how far each type of molecule will migrate through the gel. Gel electrophoresis has applications in separating DNA, RNA, proteins, and other biomolecules.
Gel electrophoresis is a method to separate biomolecules like DNA, RNA, and proteins based on their size and charge. During gel electrophoresis, charged molecules are placed in wells in a gel and an electric current is applied, causing them to migrate through the gel at different rates depending on their size and charge. Larger molecules migrate more slowly through the gel pores than smaller molecules. This allows separation of molecules by size. Common gels used include agarose and polyacrylamide. Samples can be visualized after electrophoresis using dyes like ethidium bromide or stains. Gel electrophoresis has applications in DNA sequencing, forensic analysis, and medical research.
Gel electrophoresis is a method for separation and analysis
of macromolecules (DNA, RNA and proteins) and
their fragments, based on their size and charge.
This document discusses gel electrophoresis, which separates biomolecules like DNA, RNA, and proteins based on their size and charge. It describes the basic principles of electrophoresis, including how mobility depends on factors like net charge and size. The key steps of gel electrophoresis are explained, such as preparing the gel, loading samples, running the current, and staining. Two-dimensional gel electrophoresis is also summarized, which separates proteins based on isoelectric focusing in the first dimension and size in the second dimension, allowing high resolution separation of thousands of proteins.
Electrophoresis is a technique used to separate charged particles like proteins by using an electric field to migrate them through a medium like filter paper or gel based on factors like their size and charge. It can be used to separate plasma proteins, lipoproteins, and immunoglobulins into distinct bands. The document discusses several electrophoresis techniques including paper, gel, SDS-PAGE, isoelectric focusing, and immunoelectrophoresis and how they are used to separate biomolecules.
it is a short ppt. on Electrophoresis. Which gives it's defination, Types of electrophoresis ; Like:- Zone electrophoresis, Isoelectric focussing, Immunoelectrophoresis. and their images.
Electrophoresis is a technique used to separate charged particles such as proteins, nucleic acids, and other macromolecules. It works by applying an electric field to move these particles through a medium such as a gel or paper based on their size and charge. There are different types of electrophoresis including gel electrophoresis, paper electrophoresis, capillary electrophoresis, and isoelectric focusing which separates particles based on their isoelectric point. Electrophoresis has many applications in fields like forensics, molecular biology, genetics, and biochemistry to analyze proteins, DNA, RNA, and other biomolecules.
Electrophoresis is a technique used to separate charged molecules such as proteins and nucleic acids by using an electric field. There are different types of electrophoresis including zone electrophoresis using paper, cellulose acetate, or gel matrices, and moving boundary electrophoresis without a supporting medium. Gel electrophoresis techniques like agarose gel electrophoresis and SDS-PAGE are commonly used. Electrophoresis finds applications in fields like clinical testing, forensic analysis, and environmental monitoring by allowing separation and characterization of biomolecules.
Electrophoresis is a technique used to separate charged molecules like proteins and nucleic acids. It works by applying an electric field to move molecules through a buffer solution or gel based on their size and charge. There are several types of electrophoresis that use different supporting media like agarose gel, polyacrylamide gel, cellulose acetate, or paper to separate molecules. Factors like pH, buffer composition, strength of electric field, and temperature influence how molecules separate during electrophoresis. It has various applications in biomedical research and clinical diagnostics.
Modern pharmaceutical analytical technique ( Electrophoresis)KhushbooKunkulol
This document provides an overview of electrophoresis, including definitions, principles, types, and applications. It discusses four main types of electrophoresis: paper electrophoresis, gel electrophoresis, capillary electrophoresis, and iso-electric electrophoresis. Paper and gel electrophoresis are described as zone electrophoresis techniques that separate components into bands on a supporting medium like paper or gel. Capillary electrophoresis allows separation in free solution within a thin capillary tube. Iso-electric electrophoresis separates molecules based on their iso-electric point, the pH where they have no net charge. The document outlines the principles, instrumentation, and applications of each electrophoresis technique.
Electrophoresis is a method used to separate charged particles such as proteins and nucleic acids based on differences in their migration speed in an electric field. There are several types of electrophoresis including agarose gel electrophoresis, SDS-PAGE, isoelectric focusing, capillary electrophoresis, and two-dimensional electrophoresis. During electrophoresis, charged particles migrate through a gel or capillary towards an electrode with the rate of migration depending on factors like the particle's charge, size, and shape.
This document provides information about electrophoresis, including its theory, principle, types, and applications. It begins with an introduction that defines electrophoresis as the migration of charged particles in an electric field. It then describes the theory behind electrophoresis, explaining how an electrostatic force is balanced by a drag force to produce particle velocity. The main types of electrophoresis are described as slab electrophoresis, capillary electrophoresis, paper electrophoresis, and gel electrophoresis. Key applications are identified as protein analysis, DNA analysis, and vaccine analysis. The document concludes by emphasizing the importance of electrophoresis for research and forensic investigations.
Electrophoresis along with its history, application and types are discussed here to give a brief yet understanding outlook to the topic explained which is an important topic for the biotechnology as well as all biological research field.
Electrophoresis is a technique used to separate charged molecules like proteins and nucleic acids based on their size and charge. It involves applying an electric field to move molecules through a medium like agarose gel or capillary. Shorter/less charged molecules move faster and are separated from longer/more charged molecules. It is used in laboratories and clinical settings to analyze biological samples and diagnose conditions.
Gel electrophoresis is a method used to separate molecules like proteins and nucleic acids based on their physical properties. It works by applying an electric field to move charged molecules through a gel medium. Key aspects include applying samples to the gel, running electrophoresis with an electric current, and visualizing separated molecules by staining and quantification using a densitometer. Gel electrophoresis has applications in forensics, genetics, and biochemistry to analyze DNA, diagnose diseases, and study proteins. It separates biomolecules based on factors like size, shape, and charge.
This document provides information about electrophoresis. It begins by defining electrophoresis as the migration of charged particles or molecules under the influence of an electric field. Some purposes of electrophoresis are to determine components in a sample, separate them, and obtain information about electrical double layers or determine molecular weights. The principle described is that charged particles migrate in an electric field at a velocity depending on factors like charge, size, shape and applied current. Different types of electrophoresis are described like paper, gel, and isolectric focusing electrophoresis. Key aspects of each technique including apparatus, sample application, detection, and preparation of gels are explained. Two-dimensional electrophoresis combining isolectric focusing and SDS-PAGE is also summarized.
This document discusses electrophoresis, which is the movement of charged particles in an electric field. It separates molecules based on their charge and size. Key factors that affect migration rate are listed. The main requirements for electrophoresis are an electrophoresis tank, electrodes, power supply, buffer, and specimens like serum or plasma. Common electrophoresis techniques described include zone electrophoresis using paper or gel, isoelectric focusing, immuno electrophoresis, and SDS-PAGE which separates based on size. Clinical applications involve using electrophoresis to analyze conditions like liver disease or infections.
electrophoresis-
principle
types
details on paper electrophoresis
cellulose acetate electrophoresis
zone electrophoresis
SDS-PAGE
iso-electric focussing gel electrophoresis
two-dimensional gel electrophoresis
pulsed gel electrophoresis
isotachophoresis
capillary electrophoresis
microchip electrophoresis
This document provides an overview of electrophoresis techniques. It discusses the basic principles of electrophoresis, including that the rate of migration depends on the charge-to-mass ratio of particles. It describes various electrophoretic techniques such as moving boundary electrophoresis, zone electrophoresis using free solution, gel, or paper methods, as well as isoelectric focusing, isotachophoresis, and capillary electrophoresis. It provides details on how each technique is performed and discusses their applications and advantages.
Electrophoresis is a separation technique that is based on the movement of charged particles in an electric field.
Electrophoresis is an analytical method of separating charged particles based on their relative mobilities in an electric field
Microchip Electrophoresis is the new talk of the town, which revolutionize the field of electrophoresis. It is shown to be an attractive tool for time & cost saving development of a separation method for complex sample mixtures. It made possible the simultaneous separation of catecholamines and their cationic metabolites.
Gel electrophoresis is a method to separate biomolecules like proteins, nucleic acids, and lipids based on their charge and size. During gel electrophoresis, an electric current is applied across a gel, causing negatively charged molecules to migrate toward the positive electrode and positively charged molecules to migrate toward the negative electrode. Smaller molecules migrate faster through the gel than larger molecules. Factors like the charge, size, and shape of molecules, as well as the electric current, gel composition, and buffer used, determine how far each type of molecule will migrate through the gel. Gel electrophoresis has applications in separating DNA, RNA, proteins, and other biomolecules.
Gel electrophoresis is a method to separate biomolecules like DNA, RNA, and proteins based on their size and charge. During gel electrophoresis, charged molecules are placed in wells in a gel and an electric current is applied, causing them to migrate through the gel at different rates depending on their size and charge. Larger molecules migrate more slowly through the gel pores than smaller molecules. This allows separation of molecules by size. Common gels used include agarose and polyacrylamide. Samples can be visualized after electrophoresis using dyes like ethidium bromide or stains. Gel electrophoresis has applications in DNA sequencing, forensic analysis, and medical research.
Gel electrophoresis is a method for separation and analysis
of macromolecules (DNA, RNA and proteins) and
their fragments, based on their size and charge.
This document discusses gel electrophoresis, which separates biomolecules like DNA, RNA, and proteins based on their size and charge. It describes the basic principles of electrophoresis, including how mobility depends on factors like net charge and size. The key steps of gel electrophoresis are explained, such as preparing the gel, loading samples, running the current, and staining. Two-dimensional gel electrophoresis is also summarized, which separates proteins based on isoelectric focusing in the first dimension and size in the second dimension, allowing high resolution separation of thousands of proteins.
This document provides an overview of electrophoresis, including its principle, working conditions, factors affecting separation, and types. Electrophoresis is an analytical technique that separates charged molecules like proteins and nucleic acids based on their movement in an electric field. It works by applying a voltage to move molecules through a buffer solution or gel support medium. The rate of migration depends on factors like the molecule's charge, size, and the electric field strength. Common electrophoresis techniques described include zone electrophoresis using paper, gels, and thin layers, as well as moving boundary methods like capillary electrophoresis.
The document discusses agarose gel electrophoresis. It begins with an introduction to electrophoresis and gel electrophoresis, explaining how molecules are separated based on size and charge through an applied electric field in a gel matrix. It then describes the basic components and process of agarose gel electrophoresis, including preparing the agarose gel, loading and running the samples, and visualizing the results to separate DNA fragments. Agarose gel electrophoresis is used to separate nucleic acids like DNA and RNA by size and analyze results like PCR products and DNA molecules.
Agarose gel electrophoresis by KK Sahu sirKAUSHAL SAHU
INTRODUCTION.
HISTORY.
PROCESS OF GEL ELECTROPHORESIS.
AGAROSE GEL ELECTROFORESIS.
POLYACRYALAMIDE GEL ELECTRIPHORESIS.
GEL CONDITION.
DENATURETION.
NATIVE.
BUFFERS.
USES.
CONCLUSION.
REFFERENCES.
Electrophoresis is a method used to separate charged molecules like DNA and RNA using an electric field. In agarose gel electrophoresis, DNA or RNA samples are placed into wells in an agarose gel and an electric current is applied, causing the charged molecules to migrate through the gel at rates dependent on their size. Smaller molecules migrate faster through the gel pores than larger molecules, resulting in size-based separation. Factors like agarose concentration, voltage, buffer composition influence separation resolution. Agarose gel electrophoresis is commonly used to analyze and separate DNA fragments between 200 bp to 20 kb in size.
Thank you for the detailed presentation on electrophoresis. I appreciate you taking the time to explain the key concepts and techniques. Please let me know if you have any other questions.
gel electrophoresis # GENETICS AND PTANT BRIDINGsumer06072001
mahabar, barmer, rajasthan
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Dr.s.s.rajpurohit
this ppt useful for GPB and biology student . mainly use in assignments in M.Sc. agriculture . this ppt free for student . my YouTube channel: S.S.rajpurohit , comment and contact me for agriculture ppts and other knowledge.
For gel electrophoresis textbook B.D.SINGH
Running of Agarose Gel Electrophoresis Practical Sabahat Ali
Electrophoresis technique used for separation of Macromolecules(DNA, Proteins & their derivatives)
Separation occur on the basis of charge to size ratio
Electrophoresis is a technique used to separate charged molecules like proteins and nucleic acids based on their size and charge. It involves applying an electric field to encourage migration of molecules through a medium like agarose gel or polyacrylamide gel. Shorter/lighter molecules migrate farther than longer/heavier ones. Factors like pH, electric field strength, and molecular properties determine how far molecules travel. Common electrophoresis techniques include agarose gel electrophoresis to separate DNA fragments, polyacrylamide gel electrophoresis to separate proteins, and pulsed field gel electrophoresis to separate very large DNA molecules. SDS-PAGE is commonly used to determine protein molecular weights. Electrophoresis has many applications in research and clinical diagnostics.
electrophoresis: types, advantages, disadvantages and applications.Cherry
Electrophoresis is a general term that describes the migration and separation of charged particles under the influence of an electric field.
The particles maybe simple ions, complex macromolecules and colloids or particulate matter- either living cells such as bacteria or inert material such as oil emulsion, droplet etc.
The pores present in the gel work like a sieve, allowing the smaller molecules to pass through more quickly and easily than the larger molecules.
Gel electrophoresis is a technique used to separate charged biomolecules like DNA, RNA, and proteins based on their size and charge. It works by placing the sample in wells in a gel and applying an electric current, causing the molecules to migrate through the gel at different rates towards the positive or negative electrode. Larger molecules are slowed down more by the gel matrix while smaller molecules pass through more easily, resulting in separation by size. Key factors that affect separation include the sample's charge and size, electric field strength, gel medium properties, and buffer composition and pH. Gel electrophoresis has many applications like DNA sequencing and protein analysis.
1. Electrophoresis is a technique used to separate charged particles like proteins and nucleic acids based on their size and charge. An electric field is applied, causing the particles to migrate through a medium like paper, gel, or liquid at different rates.
2. Gel electrophoresis involves applying a sample to a gel and running an electric current to separate particles based on size. Agarose and polyacrylamide gels are commonly used media that act as molecular sieves.
3. Paper electrophoresis can separate particles like proteins, but gel electrophoresis provides better resolution. Gel electrophoresis is widely used in research and clinical applications to analyze DNA, proteins, and other biomolecules.
Electrophoresis and its types and its importance in Genetic engineeringSwaatiSharma2
This document discusses electrophoresis, which is a technique used to separate biomolecules like DNA, RNA, and proteins based on their size and charge. It moves through two common media - agarose gel and polyacrylamide gel. Agarose gel is used for larger molecules while polyacrylamide gel provides better resolution for smaller molecules. The document explains the principles, factors affecting separation, different types like agarose gel electrophoresis and polyacrylamide gel electrophoresis. It also discusses the requirements, steps involved and applications of agarose and polyacrylamide gel electrophoresis.
Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge.
Different types of electrophoresis.
Gel electrophoresis; Agarose Gel electrophoresis; polyacrylamide gel electrophoresis; pulsed-field gel electrophoresis
Electrophoresis is a technique used to separate biomolecules like DNA, RNA, or proteins based on their size and charge. It works by applying an electric current to move the molecules through a gel or medium. Smaller molecules move faster through the pores in the gel than larger molecules, allowing separation. There are different types of electrophoresis that use different gel materials like agarose or polyacrylamide gels and techniques like pulsed field gel electrophoresis to separate different sized molecules. Electrophoresis is widely used in areas like molecular biology, genetics, and clinical testing.
Similar to Prabhakar singh ii sem-paper v-electrophoresis (20)
Computers are now used almost everywhere from banks to homes. A computer is an electronic device that processes raw input data into useful output information. It consists of various components including hardware, software, and input/output devices. Hardware refers to the physical and tangible parts of a computer like processors, monitors, keyboards. Software includes computer programs and operating systems. Computers use binary numbers and arithmetic for processing. Programming allows users to provide computers with instructions to perform tasks.
Catalysis is the process by which a catalyst increases or decreases the rate of a chemical reaction without itself being consumed in the process. A catalyst remains chemically unchanged at the end of the reaction. Berzelius first coined the term "catalysis" in 1836 to describe substances that increase reaction rates by loosening the bonds between reacting molecules. Catalysts can either increase (positive catalysis) or decrease (negative catalysis) reaction rates. Characteristics of catalytic reactions include that the catalyst remains unchanged after the reaction, small amounts of catalyst are effective, finely divided catalysts work best, catalysts act specifically on certain reactions but cannot initiate new ones, and changing temperature can alter reaction rates both with and without a catalyst present
1. The document discusses theories of chemical kinetics including collision theory and the effect of temperature on reaction rates.
2. It introduces the Arrhenius equation and the concept of activation energy, which is the minimum energy required for a reaction to occur.
3. Collision theory states that molecules must collide with sufficient energy and proper orientation to overcome the activation energy barrier for an effective reaction.
This document discusses various experimental methods used to study chemical kinetics, including conductometry, potentiometry, optical methods like spectrophotometry and polarimetry. It focuses on spectrophotometry, explaining how it works based on Beer's law which states that the amount of light absorbed is dependent on the concentration and path length of the absorbing substance. It describes how a spectrophotometer can be used to measure reaction rates by monitoring changes in absorbance of reactants or products over time at specific wavelengths.
1. The document discusses chemical kinetics and the factors that influence the rate of chemical reactions, such as concentration, temperature, pressure, solvent, light, and catalysts.
2. It explains concepts like the rate of reactions, rate laws, reaction orders (zero order, first order, second order), half life, and mean life. Radioactive decay is given as an example of a first order reaction.
3. The main factors that can affect the rate of a reaction are identified as the nature of the reaction, concentration of reactants, phase and surface area of reactants, temperature, pressure, presence of catalysts, type of solvent, and electromagnetic radiation.
1) The document discusses different types of colloidal states including emulsions, which are liquids dispersed in other liquids, and gels, which are liquids dispersed in solids.
2) Emulsions are unstable and require an emulsifying agent like soap to create a stable emulsion by reducing the tendency of the dispersed liquid droplets to coalesce.
3) The addition of electrolytes can cause the precipitation of colloidal particles by neutralizing their charge through adsorption of oppositely charged ions according to Hardy-Schulze rule where precipitation increases with higher valent ions.
The document discusses colloidal states and defines colloids as systems with particle sizes between 10-2000 Angstroms. It notes that Thomas Graham first classified substances into crystalloids and colloids based on their ability to diffuse through membranes. Later, it was realized that any substance can be made colloidal by subdividing particles to the appropriate size range. The document then discusses various properties of colloidal solutions or sols, including kinetic, optical, electrical properties. It also covers concepts like protective action, stability of colloids, and Hardy-Schulze law which states that precipitation increases with ion valence.
The document discusses key concepts regarding solid states and crystallography. It defines crystalline and amorphous solids, and describes the ordered crystal lattice structure of crystalline solids. It then outlines three fundamental laws of crystallography: 1) the law of constancy of interfacial angles, 2) Hauy's law of rationality of indices, and 3) the law of constancy of symmetry. The document also discusses techniques for determining crystal structures, including Bragg's law of diffraction and X-ray diffraction methods like the Laue method and powder method.
This document provides information about liquid crystals and their structure and properties. It discusses different types of liquid crystals including thermotropic, lyotropic, and metallotropic phases. Thermotropic liquid crystals exhibit phase transitions based on temperature, while lyotropic phases transition based on both temperature and concentration. Common liquid crystal phases described include nematic, smectic, chiral, and discotic phases. The document also discusses the design of liquid crystalline materials and methods for analyzing mesophases including thermal optical microscopy and differential scanning calorimetry. Biological membranes are noted to exhibit lyotropic liquid crystalline properties.
The document discusses key concepts in physical chemistry related to gaseous states including:
1) The postulates of the kinetic molecular theory of gases and deviations from ideal gas behavior.
2) Critical phenomena and real gas behavior based on the van der Waals equation of state including relationships between critical constants and van der Waals constants.
3) Maxwell's distribution of molecular velocities and concepts like root mean square, average, and most probable velocities.
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1) The growth cycle phases - lag (adaptation), log (exponential growth), and plateau (confluence with reduced growth).
2) Plating efficiency is a measure of colony formation from single cells and survival at low densities.
3) Cell synchronization techniques make cells progress through the cell cycle simultaneously to study progression.
4) Senescent cells stop dividing after 30-60 population doublings and can be identified by assays like
This document discusses techniques for primary cell culture, including mechanical disaggregation, enzymatic disaggregation using trypsin or collagenase, and the primary explant technique. It provides details on each technique, such as how tissues are chopped or sliced, the use of enzymes at different temperatures, and factors that promote efficient development of primary cultures. The document also covers criteria for cell separation methods, cell differentiation, methods of cell transformation, and characteristics of transformed cells.
1. Cell culture media must provide nutrients to support cell growth, maintain physiological pH and osmolality, and be sterile and isotonic. Early media used natural fluids but now artificial media are more common.
2. Complete media contain defined components like amino acids, vitamins, salts, glucose and organic supplements as well as serum which provides growth factors and nutrients. Different cell types require different optimized media.
3. Key properties of good culture media include maintaining pH of 7.0-7.4, appropriate levels of oxygen, carbon dioxide, and temperature usually 37°C. Osmolality is typically 260-320 mosm/kg to match cells.
The document discusses disorders of carbohydrate and lipid metabolism. It begins by defining diabetes mellitus as a metabolic disease characterized by hyperglycemia. It then describes the two main types: insulin-dependent diabetes mellitus (IDDM), which results from destruction of insulin-producing beta cells and requires insulin treatment; and non-insulin dependent diabetes mellitus (NIDDM), which is more common and often associated with obesity. It further discusses disorders like glycogen storage diseases, pentosuria, galactosemia, and regulation of blood glucose levels. Lipid disorders like hyperlipidemia are also briefly mentioned.
This document discusses disorders of lipids and lipoproteins. It begins by defining lipoproteins as complexes of lipids and proteins that transport lipids in circulation, including chylomicrons, VLDL, LDL, HDL, and fatty acid-albumin complexes. It then discusses the classification and metabolism of lipoproteins, focusing on the role of LCAT and HDL in reverse cholesterol transport. The document goes on to summarize disorders related to sphingomyelins, glycolipids, cerebrosides, cholesterol, fatty liver, and ketone bodies.
This document discusses several inborn errors of metabolism including phenylketonuria (PKU), alkaptonuria, tyrosinosis, and albinism. PKU results from a defect in the enzyme phenylalanine hydroxylase, which converts the essential amino acid phenylalanine to tyrosine in the liver. This causes phenylalanine to accumulate and be excreted as the keto acid phenylpyruvate, giving PKU its name. Alkaptonuria is due to the inability to break down tyrosine and phenylalanine, causing homogentisic acid to accumulate and deposit in connective tissues, giving them a dark color. Tyrosinosis involves a
1. The document discusses two metabolic disorders: Maple Syrup Urine Disease and Sickle Cell Anemia.
2. Maple Syrup Urine Disease is caused by a defect in the enzyme branched chain D-keto acid dehydrogenase, resulting in a buildup of amino acids and their keto acids in the blood and urine. This gives the urine a distinctive maple syrup smell.
3. Sickle Cell Anemia results from a genetic mutation that causes hemoglobin to polymerize and distort red blood cells into a sickle shape under conditions of low oxygen. The polymerization occurs due to sticky patches that form on the deoxygenated hemoglobin, binding it together into long fibers.
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This document discusses several diagnostic enzymes used in biochemical testing and disease diagnosis. It provides details on SGPT and SGOT which are used to diagnose liver damage. It also discusses aldolase which can indicate muscle or liver damage if elevated. Higher levels of aldolase may be due to various health conditions affecting the liver, muscles, or other tissues. The document also summarizes information about cholinesterase which is involved in the metabolism of certain anesthetics, and may be deficient in some people, increasing risks. Finally, it briefly mentions several other diagnostic enzymes including lactate dehydrogenase, creatine phosphokinase, alkaline phosphatase, and alcohol dehydrogenase.
1. The document discusses the functions of the kidney and pancreas, including homeostasis, excretion of waste, hormone production, and digestion.
2. Tests of pancreatic function include measuring the enzymes amylase and lipase in serum, which are elevated in conditions like pancreatitis.
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Prabhakar singh ii sem-paper v-electrophoresis
1. TEJASVI NAVADHITAMASTU
“Let our (the teacher and the taught) learning be radiant”
Let our efforts at learning be luminous and filled with joy, and
endowed with the force of purpose
Paper V: Instrumentation and Analytical Techniques
ELECTROPHORESIS
3. The movement of charged particles(ions) in an electric field resulting
in their migration towards the oppositely charged electrode is known
as electrophoresis.
Molecules with a net positive charge (cations) move towards the
negative cathode while those with net negative charge (anions)
migrate towards positive anode.
Electrophoresis is a widely used analytical technique for the
separation of biological molecules such as plasma proteins,
lipoproteins and immunoglobulins.
The rate of migration of ions in an electric field depends on several
factors that include shape, size, net charge and solvation of the ions,
viscosity of the solution and magnitude of the current employed.
ELECTROPHORESIS
4. Principle of Electrophoresis
1. Under the influence of an electric field these charged particles will migrate either to the
cathode or to the anode, depending on the nature of their net charge. When a
potential difference (voltage) is applied across the electrodes, it generates a potential
gradient, E = V/d (V= Applied Voltage, d = Distance between the electrodes).
2. When this potential gradient E is applied, the force on a molecule bearing a charge of q
coulombs is, F = Eq newtons
3. It is this force that drives a charged molecule towards an electrode. However, there is
also a frictional resistance that retards the movement of this charged molecule. This
frictional force is a measure of the hydrodynamic size of the molecule, the shape of
the molecule, the pore size of the medium in which electrophoresis is taking place and
the viscosity of the buffer. Velocity will be
4. Where f is the frictional coefficient. More commonly the term electrophoretic mobility
(m) of an ion is used, which is the ratio of the velocity of the ion to field strength (n/E).
5. When a potential difference is applied, therefore, molecules with different overall
charges will begin to separate based on different electrophoretic mobilities. Even
molecules with similar charges will begin to separate if they have different molecular
sizes, since they will experience different frictional forces.
5. 4- IMMUNOELECTROPHORESIS
1- MOVING BOUNDARY ELECTROPHORESIS
2- ZONE ELECTROPHORESIS : [A]- Paper Electrophoresis , [B] Gel Electrophoresis
3- ISOELECTRIC FOCUSSING
DIFFERENT TYPES OF ELECTROPHORESIS
Among the electrophoretic techniques, zone electrophoresis (paper,
gel), isoelectric focusing and immunoelectrophoresis are important and
commonly employed in the laboratory.
6. The original moving boundary electrophoresis, developed by
Tiselius (1933), is less frequently used these days.
In this technique, the U-tube is filled with protein solution overlaid
by a buffer solution.
As the proteins move in solution during electrophoresis, they form
boundaries which can be identified by refractive index.
1- MOVING BOUNDARY ELECTROPHORESIS
2- ZONE ELECTROPHORESIS :
A simple and modified method of moving boundary electrophoresis is the
zone electrophoresis.
An inert supporting material such as paper or gel are used.
It is geneally of two type based on supportive material –
[A]- Paper Electrophoresis , [B] Gel Electrophoresis
7. Electrophoresis of plasma proteins
In this technique, the sample is applied
on a strip of filter paper wetted with
desired buffer solution. The ends of the
strip are dipped into the buffer
reservoirs having electrodes.
The electric current is applied allowing
the molecules to migrate sufficiently.
The paper is removed, dried and
stained with a dye that specifically
colours the substances to be detected.
The coloured spots can be identified by
comparing with a set of standards run
simultaneously.
For the separation of serum proteins,
Whatman No. 1 filter paper, veronal or
tris buffer at pH 8.6 and the stains
amido black or bromophenol blue are
employed.
The serum proteins are separated into
five distinct bands—albumin, D1-, D2-,
E- and J-globulins.
[A]-PAPER ELECTROPHORESIS
8. This technique involves the separation of molecules based on their size, in
addition to the electrical charge.
The movement of large molecules is slow in gel electrophoresis (this is in
contrast to gel filtration).
The resolution is much higher in this technique.
Thus, serum proteins can be separated to about 15 bands, instead of 5 bands
on paper electrophoresis.
The gels commonly used in gel electrophoresis are agarose and
polyacrylamide, sodium dodecyl sulfate (SDS).
Polyacrylamide is employed for the determination of molecular weights of
proteins in a popularly known electrophoresis technique known as SDS-PAGE
[B]-GEL ELECTROPHORESIS
9. AGAROSE GEL ELECTROPHORESIS
The migration of the DNA molecules through the pores of the matrix must play
an important role in molecular-weight separations since the electrophoretic
mobility of DNA in free solution is independent of molecular weight.
An agarose gel is a complex network of polymeric molecules whose average
pore size depends on the buffer composition and the type and concentration of
agarose used. DNA movement through the gel was originally thought to
resemble the motion of a snake (reptation). However, real-time fluorescence
microscopy of stained molecules undergoing electrophoresis has revealed more
subtle dynamics (Schwartz & Koval 1989, Smith et al. 1989).
DNA molecules display elastic behavior by stretching in the direction of the
applied field and then contracting into dense balls. The larger the pore size of
the gel, the greater the ball of DNA which can pass through and hence the
larger the molecules which can be separated.
Once the globular volume of the DNA molecule exceeds the pore size, the DNA
molecule can only pass through by reptation. This occurs with molecules about
20 kb in size and it is difficult to separate molecules larger than this without
recourse to pulsed electrical fields
11. Properties of agarose gel
1. Agarose gel is a three-dimensional matrix formed of helical agarose molecules in
supercoiled bundles that are aggregated into three-dimensional structures with
channels and pores through which biomolecules can pass.
2. The 3-D structure is held together with hydrogen bonds and can therefore be
disrupted by heating back to a liquid state. The melting temperature is different from
the gelling temperature, depending on the sources, agarose gel has a gelling
temperature of 35-42 °C and a melting temperature of 85-95 °C. Low-melting and
low-gelling agaroses made through chemical modifications are also available.
3. The pore size of a 1% gel has been estimated from 100 nm to 200-500 nm, and its
gel strength allows gels as dilute as 0.15% to form a slab for gel electrophoresis. Low-
concentration gels (0.1 - 0.2%) however are fragile and therefore hard to handle.
4. Agarose gel has lower resolving power than polyacrylamide gel for DNA but has a
greater range of separation, and is therefore used for DNA fragments of usually 50-
20,000 bp in size. The limit of resolution for standard agarose gel electrophoresis is
around 750 kb, but resolution of over 6 Mb is possible with pulsed field gel
electrophoresis (PFGE) A 0.9% agarose gel has pores large enough for the entry
of bacteriophage T4.
12.
13.
14.
15. ELECTROENDOSMOSIS (EEO)
The agarose polymer contains charged groups, in
particular pyruvate and sulphate. These negatively charged groups create a flow
of water in the opposite direction to the movement of DNA in a process
called electroendosmosis (EEO), and can therefore retard the movement of DNA
and cause blurring of bands.
Higher concentration gel would have higher electroosmotic flow. Low EEO
agarose is therefore generally preferred for use in agarose gel electrophoresis of
nucleic acids, but high EEO agarose may be used for other purposes.
The lower sulphate content of low EEO agarose, particularly low-melting point
(LMP) agarose, is also beneficial in cases where the DNA extracted from gel is to
be used for further manipulation as the presence of contaminating sulphates
may affect some subsequent procedures, such as ligation and PCR.
Zero EEO agaroses however are undesirable for some applications as they may
be made by adding positively charged group and such groups can affect
subsequent enzyme reactions
16. MIGRATION OF NUCLEIC ACIDS IN AGAROSE GEL
A number of factors can affect the migration of nucleic acids:
1. the dimension of the gel pores (gel concentration),
2. size of DNA being electrophoresed,
3. the voltage used,
4. the ionic strength of the buffer
5. the concentration of intercalating dye such as ethidium bromide if used
during electrophoresis
For standard agarose gel electrophoresis, larger molecules are resolved better using
a low concentration gel while smaller molecules separate better at high
concentration gel. High concentrations gel however requires longer run times
The movement of the DNA may be affected by the conformation of the DNA
molecule, for example, supercoiled DNA usually moves faster than relaxed DNA
because it is tightly coiled and hence more compact. In a normal plasmid DNA
preparation, multiple forms of DNA may be present
Ethidium bromide which intercalates into circular DNA can change the charge,
length, as well as the superhelicity of the DNA molecule, therefore its presence in
gel during electrophoresis can affect its movement.
17. Migration anomalies
"Smiley" gels - this edge effect is caused when the voltage applied is too high
for the gel concentration used.
Overloading of DNA - slows down the migration of DNA fragments.
Contamination - presence of impurities, such as salts or proteins can affect the
movement of the DNA
Thermal effects: Most of the power generated during electrophoresis is
dissipated as heat. Heating of electrophoretic medium has effects as:
1. An increased rate of diffusion of sample and buffer ions leading to
broadening of the separated samples i.e. smiley band of DNA
2. The formation of convection currents, which leads to mixing of separated
samples.
3. Thermal instability of samples that are rather sensitive to heat. This may
include denaturation of proteins (and thus the loss of enzyme activity).
4. A decrease of buffer viscosity, and hence a reduction in the resistance of
the medium.
18. Mechanism of migration and separation
The negative charge of its phosphate backbone moves the DNA towards
the positively charged anode during electrophoresis.
However, the migration of DNA molecules in solution, in the absence of a
gel matrix, is independent of molecular weight during electrophoresis.
The gel matrix is therefore responsible for the separation of DNA by size
during electrophoresis
A widely accepted one is the Ogston model which treats the polymer
matrix as a sieve. A globular protein or a random coil DNA moves
through the interconnected pores, and the movement of larger
molecules is more likely to be impeded and slowed down by collisions
with the gel matrix, and the molecules of different sizes can therefore be
separated in this sieving process
19. General procedure
1. Casting of gel: The concentration of gel affects the resolution of DNA separation. For a standard
agarose gel electrophoresis, a 0.8% gives good separation or resolution of large 5–10kb DNA
fragments, while 2% gel gives good resolution for small 0.2–1kb fragments.
2. Loading of samples: The loading buffer contains a dense compound, which may be glycerol,
sucrose, or Ficoll, that raises the density of the sample so that the DNA sample may sink to the
bottom of the well. If the DNA sample contains residual ethanol after its preparation, it may float
out of the well. The loading buffer also include colored dyes such as xylene
cyanol and bromophenol blue used to monitor the progress of the electrophoresis.
3. Electrophoresis: Agarose gel electrophoresis is most commonly done horizontally in a submarine
mode whereby the slab gel is completely submerged in buffer during electrophoresis.
4. Since DNA is not visible in natural light, the progress of the electrophoresis is monitored using
colored dyes. Xylene cyanol (light blue color) comigrates large DNA fragments, while Bromophenol
blue (dark blue) comigrates with the smaller fragments. Less commonly used dyes include Cresol
Red and Orange G which migrate ahead of bromophenol blue.
5. A DNA marker is also run together for the estimation of the molecular weight of the DNA
fragments. Note however that the size of a circular DNA like plasmids cannot be accurately gauged
using standard markers unless it has been linearized by restriction digest, alternatively a
supercoiled DNA marker may be used.
6. Staining and visualization: DNA as well as RNA are normally visualized by staining with ethidium
bromide, which intercalates into the major grooves of the DNA and fluoresces under UV light. The
ethidium bromide may be added to the agarose solution before it gels, or the DNA gel may be
stained later after electrophoresis. Destaining of the gel is not necessary but may produce better
images. Other methods of staining are available; examples are SYBR Green, GelRed, methylene
blue, brilliant cresyl blue, Nile blue sulphate, and crystal violet
20. Buffers
In general, the ideal buffer should have good conductivity, produce
less heat and have a long life.[29] There are a number of buffers
used for agarose electrophoresis; common ones for nucleic acids
include Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE).
Tris-phosphate buffer has high buffering capacity but cannot be
used if DNA extracted is to be used in phosphate sensitive
reaction.
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.
21. Application of agarose gel electrophoresis
1. Estimation of the size of DNA molecules following restriction enzyme digestion, e.g.
in restriction mapping of cloned DNA.
2. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
3. Separation of DNA fragments for extraction and purification.
4. Separation of restricted genomic DNA prior to Southern transfer, or of RNA prior
to Northern transfer.
5. Agarose gels are easily cast and handled compared to other matrices and nucleic
acids are not chemically altered during electrophoresis. Samples are also easily
recovered. After the experiment is finished, the resulting gel can be stored in a
plastic bag in a refrigerator.
6. 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.
22. POLYACRYLAMIDE GEL ELECTROPHORESIS
PAGE is used for separating proteins in size from 5 to 2,000 kDa due to the uniform
pore size in polyacrylamide gel. Pore size is controlled by modulating the
concentrations of acrylamide and bis-acrylamide powder used in creating a gel.
Traditional DNA sequencing techniques such as Maxam-Gilbert or Sanger methods
used polyacrylamide gels to separate DNA fragments differing by a single base-pair
in length so the sequence could be read. Most modern DNA separation methods
now use agarose gels, except for particularly small DNA fragments. It is currently
most often used in the field of immunology and protein analysis, often used to
separate different proteins or isoforms of the same protein into separate bands.
Typically resolving gels are made in 6%, 8%, 10%, 12% or 15%. Stacking gel (5%) is
poured on top of the resolving gel and a gel comb (which forms the wells and
defines the lanes where proteins, sample buffer and ladders will be placed) is
inserted. The percentage chosen depends on the size of the protein that one
wishes to identify or probe in the sample. The smaller the known weight, the
higher the percentage that should be used. Changes on the buffer system of the
gel can help to further resolve proteins of very small sizes
32. IMMUNOELECTROPHORESIS
This technique involves combination of the principles of electrophoresis and
immunological reactions. Immunoelectrophoresis is useful for the analysis of
complex mixtures of antigens and antibodies.
The complex proteins of biological samples (say human serum) are subjected
to electrophoresis. The antibody (antihuman immune serum from rabbit or
horse) is thenapplied in a trough parallel to the electrophoretic separation.
The antibodies diffuse and, when they come in contact with antigens,
precipitation occurs, resulting in the formation of precipitin bands which can
be identified
33. ISOTACHOPHORESIS
Isotachophoresis (ITP) is a technique in analytical chemistry used for selective
separation and concentration of ionic analytes. It is a form of electrophoresis:
charged analytes are separated based on ionic mobility, a factor which tells how fast
an ion migrates through an electric field.
Isotachophoresis means migration with the same speed. Many "non physisists"
complain, that they do not understand isotachophoresis as easily as other biophysical
methods. The reason: several things occur simultaneously.
To understand the effects and the features of the technique, one should imagine the
four facts of isotachophoresis, which are equally important and which happen at the
same time:
1. Migration of all ions with the same speed
2. Separation of components as an "Ion train"
3. Zone sharpening effect
4. Concentration regulating effect
34. In isotachophoresis (ITP), the separation is carried out in a discontinuous
buffer system. The ionized sample migrates between a leading electrolyte
with a high mobility and a terminating - sometimes called trailing - ion with a
low mobility, all of them migrating with the same speed.
The different components are separated according to their electrophoretic
mobilities and form stacks: the substance with the highest mobility directly
follows the leading ion, the one with the lowest mobility migrates directly in
front of the terminating electrolyte.
In ITP there is a concentration regulating effect which works against diffusion.
In an anionic separation, the leading electrolyte will be at the anodal, and the
terminating electrolyte at the cathodal side. The sample is applied between
the two. The system also contains a common cationic counter-ion.
A practical example: Chloride is the leading ion, Glycine is the terminating
ion, Tris is the counter ion.
ITP is mostly applied for stacking of the samples during the first phase of disc
electrophoresis.
35. PULSED-FIELD GEL ELECTROPHORESIS
At Columbia University in 1984, David C. Schwartz and Charles Cantor developed
a variation on the standard protocol by introducing an alternating voltage
gradient to improve the resolution of larger molecules. This technique became
known as pulsed-field gel electrophoresis (PFGE).
Pulsed field gel electrophoresis is a technique used for the separation of
large deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an
electric field that periodically changes direction.
Standard gel electrophoresis techniques for separation of DNA molecules
provided huge advantages for molecular biology research. However, it was
unable to separate very large molecules of DNA effectively.
DNA molecules larger than 15-20 kb migrating through a gel will essentially
move together in a size-independent manner. The development of PFGE
expanded the range of resolution for DNA fragments by as much as two orders
of magnitude.
36. PROCEDURE
The procedure for this technique is relatively similar to performing a
standard gel electrophoresis except that instead of constantly running the
voltage in one direction, the voltage is periodically switched among three
directions; one that runs through the central axis of the gel and two that
run at an angle of 60 degrees either side.
The pulse times are equal for each direction resulting in a net forward
migration of the DNA. For extremely large molecules (up to around 2 Mb),
switching-interval ramps can be used that increases the pulse time for
each direction over the course of a number of hours—take, for instance,
increasing the pulse linearly from 10 seconds at 0 hours to 60 seconds at
18 hours.
This procedure takes longer than normal gel electrophoresis due to the
size of the fragments being resolved and the fact that the DNA does not
move in a straight line through the gel.
37. THEORY
While in general small fragments can find their way through
the gel matrix more easily than large DNA fragments, a
threshold length exists above 30–50 kb where all large
fragments will run at the same rate, and appear in a gel as a
single large diffuse band.
However, with periodic changing of field direction, the various
lengths of DNA react to the change at differing rates. That is,
larger pieces of DNA will be slower to realign their charge
when field direction is changed, while smaller pieces will be
quicker. Over the course of time with the consistent changing
of directions, each band will begin to separate more and more
even at very large lengths. Thus separation of very large DNA
pieces using PFGE is made possible
38. 1. PFGE may be used for genotyping or genetic fingerprinting. It is commonly
considered a gold standard in epidemiological studies of pathogenic
organisms. Subtyping has made it easier to discriminate among strains
of Listeria monocytogenes and thus to link environmental or food isolates
with clinical infections.
2. PFGE allows investigators to separate much larger pieces of DNA than
conventional agarose gel electrophoresis. In conventional gels, the current is
applied in a single direction (from top to bottom). But in PFGE, the direction of
the current is altered at a regular interval as shown in the animated gif below.
3. The gray box is the gel, the six sets of 4 black lines represent the 3 pairs of
electrodes. Initially, the gel is empty but soon Whole chromosomes mixed
with blue loading dye will be placed into the wells. Then the current will be
turned on and the direction of the current will change in a regular pattern.
This is repeated until the loading dye reaches near the end of the gel Then the
gel is soaked in a solution containing ethidium bromide which fluoresces
orange when bound to DNA.
APPLICATIONS
39. CONTOUR-CLAMPED HOMOGENEOUS ELECTRICAL-
FIELD (CHEF) ELECTROPHORESIS
A major disadvantage of PFGE, as originally described, is that the samples do not
run in straight lines. This makes subsequent analysis difficult. This problem has
been overcome by the development of improved methods for alternating the
electrical field. The most popular of these is contour-clamped homogeneous
electrical-field (CHEF) electrophoresis (Chu et al.1986).
In early CHEF-type systems the
reorientation angle was fixed at
120°. However, in newer systems,
the reorientation angle can be
varied and it has been found that for
whole-yeast chromosomes the
migration rate is much faster with
an angle of 106°.
Fragments of DNA as large as 200–
300 kb are routinely handled in
genomics work and these can be
separated in a matter of hours using
CHEF systems with a reorientation
angle of 90° or less.
40. OFFGEL electrophoresis
OFFGEL electrophoresis separates proteins or peptides according to
their isoelectric points, whereby the separated components are
recovered in liquid phase. Up- or downstream sample processing
steps such as immunodepletion, protein digestion and liquid
chromatography can be easily interfaced with this technique for
multi-dimensional separations of complex samples.
With its new state-of-the-art web-based user interface OFFGEL
electrophoresis has just become even more intuitive to use.
Directly connect it to the supplied Netbook or connect to your local
area network to operate from any PC* or iPhone
41. OFFGEL ELECTROPHORESIS – High resolution pI fractionation
based on proven IPG technology
The Agilent 3100 OFFGEL Fractionator performs isoelectric focusing of proteins or
peptides in immobilized pH gradient (IPG) gel strips. The technique applied by the
3100 OFFGEL Fractionator differs from conventional gel electrophoresis in that
sample components are recovered from liquid phase. This makesthe technology
directly compatible with liquid-phase workflows, for example LC-MS.
1. The IPG gel strip is rehydrated and seals tightly against the well frame.
2. The diluted sample is equally distributed into all wells in the frame, and a cover
seal is placed on top of the frame to prevent sample evaporation.
3. A high voltage is applied to the ends of the gel strip and the protein or peptide
molecules migrate through the gel until they reach a position where the pH
equals the pI of the molecule.
4. After fractionation, the separated proteins or peptides remain in the buffer
solution and can be easily removed using a pipette for subsequent downstream
processing.