This document provides an overview of electrophoresis techniques. It discusses different types of electrophoresis including moving boundary electrophoresis, zone electrophoresis, gel electrophoresis, and capillary electrophoresis. Agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) are described in more detail. The document outlines the basic principles, components, sample preparation, running, and visualization of electrophoresis gels.
Paper electrophoresis is a technique used to separate charged molecules like amino acids, peptides, and proteins. It works by applying an electric current across a paper strip soaked in buffer solution, which causes the charged molecules in a sample to migrate across the paper at different rates based on their charge and size. The document discusses the principle, requirements, procedure, advantages, types (based on voltage and design), and factors affecting migration of molecules in paper electrophoresis. It is a simple, inexpensive, and widely-used method to qualitatively analyze biological samples for clinical diagnosis and research purposes.
Gel electrophoresis is a technique used to separate molecules like proteins and nucleic acids based on their size and charge. It works by applying an electric current to a gel, which causes negatively charged molecules to migrate toward the positive electrode at rates dependent on their size and charge. There are different types of gels used like agarose, polyacrylamide, and starch. Agarose is commonly used for separating DNA fragments, while polyacrylamide is used for separating smaller molecules like proteins and nucleic acids. The separated molecules can then be visualized by staining the gel. Gel electrophoresis is widely applied in areas like determining protein purity and molecular weights.
This document discusses capillary electrophoresis, a technique for separating charged molecules. It can separate proteins, peptides, amino acids, nucleic acids, and other molecules. Capillary electrophoresis works by applying an electric field across a thin capillary tube, which causes different molecules to migrate through the buffer at different rates based on their charge and size. It provides high separation efficiency using only small sample volumes. The document outlines the basic components and process of capillary electrophoresis.
Vertical Gel Electrophoresis (SDS-PAGE)Srikanth H N
Vertical gel electrophoresis has several advantages over horizontal gel electrophoresis. It allows for the use of a discontinuous buffer system to separate proteins, which is not possible with horizontal gels. The technique involves pouring an acrylamide gel between glass plates to a thickness of less than 2 mm. Samples are loaded and subjected to an electric current, with cations moving toward the cathode and anions toward the anode. Proteins are separated based on their size and charge using techniques like SDS-PAGE, which involves denaturing proteins to impart a uniform charge.
In this slide contains introduction, methods, supporting media for zone electrophoresis.
Presented by: Mary Vishali. (Department of pharmacology),
RIPER, anantapur.
Capillary electrophoresis principles and applications Indira Shastry
Capillary electrophoresis is a technique used to separate charged molecules like proteins, nucleic acids, and other small molecules based on their electrophoretic mobility. It has several advantages over traditional gel electrophoresis methods like faster separation, higher resolution, and requiring only small sample volumes. In capillary electrophoresis, samples are injected into a thin, fused silica capillary tube and separated under the influence of an applied electric field based on differences in their charge and size. Key applications of capillary electrophoresis include hemoglobin electrophoresis for detecting abnormal hemoglobins, serum protein electrophoresis, and DNA sequencing. It provides a rapid, automated, and high-resolution method for analyzing biomolecules.
Paper electrophoresis is a technique used to separate charged molecules like amino acids, peptides, and proteins. It works by applying an electric current across a paper strip soaked in buffer solution, which causes the charged molecules in a sample to migrate across the paper at different rates based on their charge and size. The document discusses the principle, requirements, procedure, advantages, types (based on voltage and design), and factors affecting migration of molecules in paper electrophoresis. It is a simple, inexpensive, and widely-used method to qualitatively analyze biological samples for clinical diagnosis and research purposes.
Gel electrophoresis is a technique used to separate molecules like proteins and nucleic acids based on their size and charge. It works by applying an electric current to a gel, which causes negatively charged molecules to migrate toward the positive electrode at rates dependent on their size and charge. There are different types of gels used like agarose, polyacrylamide, and starch. Agarose is commonly used for separating DNA fragments, while polyacrylamide is used for separating smaller molecules like proteins and nucleic acids. The separated molecules can then be visualized by staining the gel. Gel electrophoresis is widely applied in areas like determining protein purity and molecular weights.
This document discusses capillary electrophoresis, a technique for separating charged molecules. It can separate proteins, peptides, amino acids, nucleic acids, and other molecules. Capillary electrophoresis works by applying an electric field across a thin capillary tube, which causes different molecules to migrate through the buffer at different rates based on their charge and size. It provides high separation efficiency using only small sample volumes. The document outlines the basic components and process of capillary electrophoresis.
Vertical Gel Electrophoresis (SDS-PAGE)Srikanth H N
Vertical gel electrophoresis has several advantages over horizontal gel electrophoresis. It allows for the use of a discontinuous buffer system to separate proteins, which is not possible with horizontal gels. The technique involves pouring an acrylamide gel between glass plates to a thickness of less than 2 mm. Samples are loaded and subjected to an electric current, with cations moving toward the cathode and anions toward the anode. Proteins are separated based on their size and charge using techniques like SDS-PAGE, which involves denaturing proteins to impart a uniform charge.
In this slide contains introduction, methods, supporting media for zone electrophoresis.
Presented by: Mary Vishali. (Department of pharmacology),
RIPER, anantapur.
Capillary electrophoresis principles and applications Indira Shastry
Capillary electrophoresis is a technique used to separate charged molecules like proteins, nucleic acids, and other small molecules based on their electrophoretic mobility. It has several advantages over traditional gel electrophoresis methods like faster separation, higher resolution, and requiring only small sample volumes. In capillary electrophoresis, samples are injected into a thin, fused silica capillary tube and separated under the influence of an applied electric field based on differences in their charge and size. Key applications of capillary electrophoresis include hemoglobin electrophoresis for detecting abnormal hemoglobins, serum protein electrophoresis, and DNA sequencing. It provides a rapid, automated, and high-resolution method for analyzing biomolecules.
Paper electrophoresis is a simple and inexpensive technique that uses filter paper and micro quantities of plasma to separate molecules like proteins, amino acids, and oligopeptides based on their charge. The basic equipment needed includes a power pack to provide a stabilized current and an electrophoretic cell containing electrodes, buffer reservoirs, and a support for the filter paper. Samples are applied to filter paper strips, which are placed in buffer solutions and an electric field is applied, causing charged particles to migrate and separate into distinct bands based on their size and charge. Paper electrophoresis is commonly used to analyze proteins in human plasma and other biological samples.
The document discusses capillary electrophoresis (CE), including its key terminology, instrumentation, flow dynamics, and factors that affect separation efficiency such as capillary diameter, voltage, and temperature. CE uses narrow capillaries to perform high-efficiency separations of charged molecules. When an electric field is applied, electroosmotic flow and electrophoretic migration move solutes through the capillary at different rates depending on their size and charge. Precise temperature control and optimization of factors like voltage and capillary diameter are important for achieving high resolution separations.
Technique combining ideas of isoelectric points and electric fields. It gives good separation with a high resolution compared to any other method. Proteins are separated in a pH gradient according to their isoelectric points, with proteins migrating towards the cathode or anode depending on whether their pI is below or above the pH at that point in the gradient. Applications include separation and identification of serum proteins in clinical settings and separation of proteins, enzymes, and other biomolecules in research.
Nuclear magnetic resonance (enzymology) Mohsin Shad
Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique used to determine the structure of organic compounds. It works by applying a magnetic field to atomic nuclei, which resonate at radio frequencies characteristic of their chemical environment. NMR can characterize very small sample amounts without destruction. The principle relies on nuclear spin and how nuclei align in an external magnetic field. NMR instrumentation includes a magnet, coils, transmitter, receiver, and computer system. Chemical shifts are measured in parts per million relative to a standard. NMR has various applications including determining biomolecular structures in solution and studying chemical and dynamic properties of functional groups.
Gel electrophoresis is a technique used to separate macromolecules like proteins and nucleic acids based on size, charge, or conformation. It works by applying an electric field to push molecules through a gel containing small pores. Smaller molecules will travel farther through the gel than larger ones. After electrophoresis, the gel can be stained to make the separated molecules visible, then used for various applications like DNA fingerprinting in forensics, mapping cellular components in biochemistry, and determining purity in protein analysis.
Electrophoresis is a technique used to separate charged biomolecules like proteins and nucleic acids. It works by applying an electric field to migrate these molecules through a gel or liquid medium based on their size and charge. There are different types of electrophoresis depending on the supporting medium used like agarose gel, polyacrylamide gel or free solution. Gel electrophoresis is commonly used and involves using a gel like agarose or polyacrylamide as a sieving matrix to separate molecules based on size. The separated molecules can then be visualized using stains and have various applications in research and clinical diagnostics.
Zone electrophoresis is an electrophoretic technique used to separate charged particles like proteins, nucleic acids, and biopolymers. It works by migrating the charged particles through a stabilizing medium like paper, agarose gel, or polyacrylamide gel under the influence of an electric field. The separated components form discrete zones on the supporting medium. Common types of zone electrophoresis include paper electrophoresis, gel electrophoresis using agarose or polyacrylamide, cellulose acetate electrophoresis, and thin layer electrophoresis. Each technique has advantages and applications for separating different types of biological molecules.
The principle and performance of capillary electrophoresisimprovemed
Capillary electrophoresis is a technique that uses thin capillary tubes to separate ionic and molecular species based on their electrophoretic mobility. Key aspects of the technique include the use of an electric field to drive electrophoretic separation and electroosmotic flow, which allows both cations and anions to be separated in a single run. Common modes of capillary electrophoresis are capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, and micellar electrokinetic chromatography. The document provides detailed explanations of the principles and instrumentation of capillary electrophoresis.
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.
Column chromatography is a technique used to separate mixtures. It uses a column packed with a stationary phase, which can be a solid or liquid. The mixture is dissolved in a mobile phase and passed through the column. Components are separated based on their differing interactions with the stationary phase. Selection of the proper stationary and mobile phases is important for successful separation. Column chromatography is useful for purifying compounds and isolating metabolites.
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 gel or other medium based on their size and charge. The document discusses the principles of electrophoresis, factors that affect separation like buffer composition and pH, and different electrophoresis techniques including paper, agarose gel, and polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE) is commonly used to separate proteins based on their size and charge to mass ratio.
Electrophoresis is a technique used to separate charged molecules such as proteins and nucleic acids. It works by applying an electric field to move these molecules through a medium such as a gel or paper. There are different types of electrophoresis based on the medium used and whether the molecules are separated by size alone or by their charge. Zone electrophoresis separates molecules into discrete zones based on their charge and size, while moving boundary electrophoresis separates molecules continuously. SDS-PAGE is a common electrophoresis method that uses SDS detergent to denature proteins and give them a uniform charge-to-size ratio for separation based only on their molecular weight.
This document provides information about electrophoresis. It discusses different types of electrophoretic techniques including slab electrophoresis, capillary electrophoresis, capillary zone electrophoresis, capillary gel electrophoresis, capillary isotachophoresis, and micellar electrokinetic chromatography. It also covers principles, instrumentation, applications in areas like DNA analysis and vaccine analysis.
Isoelectric focusing is a technique used to separate proteins based on their isoelectric point. It involves creating an immobilized pH gradient using carrier ampholytes within an acrylamide gel. When an electric current is applied, proteins will migrate within the gel until they reach the point where they carry no net charge and stop, allowing separation based on subtle differences in pI. The key steps are preparation of the IEF gel, addition of ampholytes to generate the pH gradient, running electrophoresis to allow protein migration, and staining to visualize the separated protein bands.
Capillary electrophoresis is a separation technique that uses narrow bore capillaries. Charged molecules migrate through the capillary under the influence of an applied electric field and separate based on their charge and size. The principle involves electrostatic forces moving molecules toward the electrode of opposite charge, as well as electroosmotic flow dragging buffer molecules. Capillary electrophoresis has various modes of operation and is used to separate and analyze biological samples in clinical and diagnostic applications.
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.
Affinity chromatography is a method to separate molecules based on specific biological interactions between a ligand attached to a support and the target molecule. It relies on reversible interactions such as those between enzymes and substrates, antibodies and antigens, or receptors and ligands. The target molecule binds to the ligand when a sample mixture is passed through the column, while other molecules pass through. The bound target molecule can then be eluted by changing conditions like pH or ionic strength to disrupt the specific interaction. Common applications of affinity chromatography include purifying antibodies, recombinant tagged proteins, and lectins.
Isoelectric focusing electrophoresis
Isoelectric-focusing electrophoresis is a type of electrophoresis. The separation technique involves electrophoresis based on the isoelectric point of the sample.
The document summarizes a seminar report presented on September 1st, 2014 by K. Swapna on the topic of electrophoresis, which was guided by Utham Prasad SIR. It discusses different types of electrophoresis techniques including moving boundary electrophoresis and zonal electrophoresis. It provides details on electrophoretic mobility and the factors that affect it. It also explains moving boundary electrophoresis and zonal electrophoresis, highlighting methods to stabilize components in zonal electrophoresis such as using support material, density gradients, and free zone electrophoresis.
SDS-PAGE electrophoresis is a technique used to separate proteins by size. It involves running proteins through a stacking gel and resolving gel with an electric current. The stacking gel concentrates the proteins into a narrow band before entering the resolving gel, which separates the proteins based on size differences. Key components of SDS-PAGE include SDS to impart identical charge-to-mass ratios to proteins, reducing agents to unfold proteins, and polyacrylamide gels which sieve proteins during electrophoresis based on their size.
Paper electrophoresis is a simple and inexpensive technique that uses filter paper and micro quantities of plasma to separate molecules like proteins, amino acids, and oligopeptides based on their charge. The basic equipment needed includes a power pack to provide a stabilized current and an electrophoretic cell containing electrodes, buffer reservoirs, and a support for the filter paper. Samples are applied to filter paper strips, which are placed in buffer solutions and an electric field is applied, causing charged particles to migrate and separate into distinct bands based on their size and charge. Paper electrophoresis is commonly used to analyze proteins in human plasma and other biological samples.
The document discusses capillary electrophoresis (CE), including its key terminology, instrumentation, flow dynamics, and factors that affect separation efficiency such as capillary diameter, voltage, and temperature. CE uses narrow capillaries to perform high-efficiency separations of charged molecules. When an electric field is applied, electroosmotic flow and electrophoretic migration move solutes through the capillary at different rates depending on their size and charge. Precise temperature control and optimization of factors like voltage and capillary diameter are important for achieving high resolution separations.
Technique combining ideas of isoelectric points and electric fields. It gives good separation with a high resolution compared to any other method. Proteins are separated in a pH gradient according to their isoelectric points, with proteins migrating towards the cathode or anode depending on whether their pI is below or above the pH at that point in the gradient. Applications include separation and identification of serum proteins in clinical settings and separation of proteins, enzymes, and other biomolecules in research.
Nuclear magnetic resonance (enzymology) Mohsin Shad
Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique used to determine the structure of organic compounds. It works by applying a magnetic field to atomic nuclei, which resonate at radio frequencies characteristic of their chemical environment. NMR can characterize very small sample amounts without destruction. The principle relies on nuclear spin and how nuclei align in an external magnetic field. NMR instrumentation includes a magnet, coils, transmitter, receiver, and computer system. Chemical shifts are measured in parts per million relative to a standard. NMR has various applications including determining biomolecular structures in solution and studying chemical and dynamic properties of functional groups.
Gel electrophoresis is a technique used to separate macromolecules like proteins and nucleic acids based on size, charge, or conformation. It works by applying an electric field to push molecules through a gel containing small pores. Smaller molecules will travel farther through the gel than larger ones. After electrophoresis, the gel can be stained to make the separated molecules visible, then used for various applications like DNA fingerprinting in forensics, mapping cellular components in biochemistry, and determining purity in protein analysis.
Electrophoresis is a technique used to separate charged biomolecules like proteins and nucleic acids. It works by applying an electric field to migrate these molecules through a gel or liquid medium based on their size and charge. There are different types of electrophoresis depending on the supporting medium used like agarose gel, polyacrylamide gel or free solution. Gel electrophoresis is commonly used and involves using a gel like agarose or polyacrylamide as a sieving matrix to separate molecules based on size. The separated molecules can then be visualized using stains and have various applications in research and clinical diagnostics.
Zone electrophoresis is an electrophoretic technique used to separate charged particles like proteins, nucleic acids, and biopolymers. It works by migrating the charged particles through a stabilizing medium like paper, agarose gel, or polyacrylamide gel under the influence of an electric field. The separated components form discrete zones on the supporting medium. Common types of zone electrophoresis include paper electrophoresis, gel electrophoresis using agarose or polyacrylamide, cellulose acetate electrophoresis, and thin layer electrophoresis. Each technique has advantages and applications for separating different types of biological molecules.
The principle and performance of capillary electrophoresisimprovemed
Capillary electrophoresis is a technique that uses thin capillary tubes to separate ionic and molecular species based on their electrophoretic mobility. Key aspects of the technique include the use of an electric field to drive electrophoretic separation and electroosmotic flow, which allows both cations and anions to be separated in a single run. Common modes of capillary electrophoresis are capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, and micellar electrokinetic chromatography. The document provides detailed explanations of the principles and instrumentation of capillary electrophoresis.
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.
Column chromatography is a technique used to separate mixtures. It uses a column packed with a stationary phase, which can be a solid or liquid. The mixture is dissolved in a mobile phase and passed through the column. Components are separated based on their differing interactions with the stationary phase. Selection of the proper stationary and mobile phases is important for successful separation. Column chromatography is useful for purifying compounds and isolating metabolites.
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 gel or other medium based on their size and charge. The document discusses the principles of electrophoresis, factors that affect separation like buffer composition and pH, and different electrophoresis techniques including paper, agarose gel, and polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE) is commonly used to separate proteins based on their size and charge to mass ratio.
Electrophoresis is a technique used to separate charged molecules such as proteins and nucleic acids. It works by applying an electric field to move these molecules through a medium such as a gel or paper. There are different types of electrophoresis based on the medium used and whether the molecules are separated by size alone or by their charge. Zone electrophoresis separates molecules into discrete zones based on their charge and size, while moving boundary electrophoresis separates molecules continuously. SDS-PAGE is a common electrophoresis method that uses SDS detergent to denature proteins and give them a uniform charge-to-size ratio for separation based only on their molecular weight.
This document provides information about electrophoresis. It discusses different types of electrophoretic techniques including slab electrophoresis, capillary electrophoresis, capillary zone electrophoresis, capillary gel electrophoresis, capillary isotachophoresis, and micellar electrokinetic chromatography. It also covers principles, instrumentation, applications in areas like DNA analysis and vaccine analysis.
Isoelectric focusing is a technique used to separate proteins based on their isoelectric point. It involves creating an immobilized pH gradient using carrier ampholytes within an acrylamide gel. When an electric current is applied, proteins will migrate within the gel until they reach the point where they carry no net charge and stop, allowing separation based on subtle differences in pI. The key steps are preparation of the IEF gel, addition of ampholytes to generate the pH gradient, running electrophoresis to allow protein migration, and staining to visualize the separated protein bands.
Capillary electrophoresis is a separation technique that uses narrow bore capillaries. Charged molecules migrate through the capillary under the influence of an applied electric field and separate based on their charge and size. The principle involves electrostatic forces moving molecules toward the electrode of opposite charge, as well as electroosmotic flow dragging buffer molecules. Capillary electrophoresis has various modes of operation and is used to separate and analyze biological samples in clinical and diagnostic applications.
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.
Affinity chromatography is a method to separate molecules based on specific biological interactions between a ligand attached to a support and the target molecule. It relies on reversible interactions such as those between enzymes and substrates, antibodies and antigens, or receptors and ligands. The target molecule binds to the ligand when a sample mixture is passed through the column, while other molecules pass through. The bound target molecule can then be eluted by changing conditions like pH or ionic strength to disrupt the specific interaction. Common applications of affinity chromatography include purifying antibodies, recombinant tagged proteins, and lectins.
Isoelectric focusing electrophoresis
Isoelectric-focusing electrophoresis is a type of electrophoresis. The separation technique involves electrophoresis based on the isoelectric point of the sample.
The document summarizes a seminar report presented on September 1st, 2014 by K. Swapna on the topic of electrophoresis, which was guided by Utham Prasad SIR. It discusses different types of electrophoresis techniques including moving boundary electrophoresis and zonal electrophoresis. It provides details on electrophoretic mobility and the factors that affect it. It also explains moving boundary electrophoresis and zonal electrophoresis, highlighting methods to stabilize components in zonal electrophoresis such as using support material, density gradients, and free zone electrophoresis.
SDS-PAGE electrophoresis is a technique used to separate proteins by size. It involves running proteins through a stacking gel and resolving gel with an electric current. The stacking gel concentrates the proteins into a narrow band before entering the resolving gel, which separates the proteins based on size differences. Key components of SDS-PAGE include SDS to impart identical charge-to-mass ratios to proteins, reducing agents to unfold proteins, and polyacrylamide gels which sieve proteins during electrophoresis based on their size.
This document provides instructions for performing electrophoresis to separate and characterize proteins. It describes how proteins are linearized and given a negative charge when mixed with SDS detergent, allowing them to be separated by size when an electric current is applied across a polyacrylamide gel medium. Detailed procedures are given for preparing the gel, loading samples mixed with buffer, running the electrophoresis, and staining proteins in the gel for visualization and analysis. A list of standard marker proteins and their molecular weights is also provided for comparison.
This document discusses two-dimensional gel electrophoresis (2DGE), a powerful technique for analyzing complex protein mixtures. 2DGE separates proteins in two steps - first by isoelectric focusing to separate proteins by their isoelectric point, followed by SDS-PAGE to separate by molecular weight. This allows thousands of proteins to be separated simultaneously on a single gel based on these two properties. The document provides details on the principles, protocols, and improvements of the 2DGE technique.
Protein Detection Methods and Applicationangelsalaman
The document discusses various protein analysis methods including gel electrophoresis, SDS-PAGE, and native gel electrophoresis. Gel electrophoresis separates proteins, DNA, and RNA using an electric current applied to a gel matrix. SDS-PAGE separates denatured proteins by size, coating them with negative charges. Native gel electrophoresis separates intact proteins by their intrinsic charge and hydrodynamic size, allowing analysis of conformation, aggregation, and binding events.
This document provides an overview of electrophoresis techniques presented by Miss Sayanti Sau. It discusses the basic principles of electrophoresis and defines different types including zone electrophoresis techniques like paper, gel, thin layer, and cellulose acetate electrophoresis. It also covers moving boundary electrophoresis techniques such as capillary electrophoresis, isotachophoresis, and isoelectric focusing. Details are provided on gel electrophoresis methods including agarose, polyacrylamide, and SDS-PAGE. Applications and advantages of various electrophoresis techniques are highlighted.
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.
Electrophoresis is a laboratory technique used to separate DNA, RNA or protein molecules based on their size and electrical charge. lectrophoresis is based on the phenomenon that most biomolecules exist as electrically-charged particles, possessing ionizable functional groups. Gel electrophoresis is widely used in the molecular biology and biochemistry labs in areas such as forensic science, conservational biology, and medicine.
This document summarizes the process of 2-D gel electrophoresis used to separate proteins. It begins with an introduction explaining that 2-D gel electrophoresis couples isoelectric focusing in the first dimension based on isoelectric point and SDS-PAGE in the second dimension based on molecular mass. The document then covers the principles, processes, and techniques involved in isoelectric focusing, SDS-PAGE, blotting, and concludes by discussing the applications and references for 2-D gel electrophoresis.
Electrophoresis is a technique used to separate macromolecules like proteins, DNA, and RNA based on their charge and size using an electric field. Ferdinand Frederic Reuss first observed electrophoresis in 1807 while working at Moscow State University. During electrophoresis, charged molecules migrate toward the electrode of opposite charge - negatively charged molecules move toward the positive electrode. There are different types of electrophoresis including gel electrophoresis, capillary electrophoresis, zone electrophoresis, and immunoelectrophoresis. Gel electrophoresis involves preparing a gel, loading samples into wells, applying an electric current to separate the molecules by size.
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 is a technique used to separate charged molecules like proteins and nucleic acids based on their size and charge. It works by applying an electric current to move these molecules through a medium like a gel or paper. The document discusses different types of electrophoresis like gel electrophoresis, paper electrophoresis, and isolectric focusing. It also explains how various factors like the molecule's charge, size, and shape affect its movement during electrophoresis.
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
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.
This document provides an overview of electrophoresis, including:
1. Electrophoresis uses the migration of charged solutes or particles in a liquid medium under the influence of an electric field. It is widely used to separate biological molecules like proteins.
2. Particles with different charge-to-mass ratios migrate at different rates depending on factors like their net charge, size, and the pH and strength of the buffer solution. Agarose gel and polyacrylamide gel electrophoresis are commonly used techniques.
3. The general procedure involves separating the particles in an electric field, staining to visualize the bands, then detecting and quantifying the separated fractions. Automated systems now allow high-throughput processing of
Electrophoresis by Anubhav Singh, M.pharmAnubhav Singh
This document provides an overview of electrophoresis. It begins by explaining how electrophoresis works, noting that when an electric field is applied to a colloidal solution, colloidal particles migrate toward either the positive or negative electrode depending on their charge. It then discusses several types of electrophoresis, including SDS electrophoresis, native gels, electrofocusing gels, and DNA agarose gels. The document also outlines the basic equipment and process for performing electrophoresis, including sample delivery, gel preparation, loading samples, running the gel, and analyzing results. It concludes by mentioning some applications of electrophoresis like DNA sequencing and medical research.
The document discusses electrophoresis techniques. It describes how electrophoresis works, with charged molecules moving through an electric field at different rates depending on their size and charge. It explains the basics of two main types - moving boundary electrophoresis, which lacks a supporting medium, and zone electrophoresis, which uses a gel or other medium to better separate molecules. It focuses on gel electrophoresis, describing how polyacrylamide gels are made and how vertical gel electrophoresis is performed. Applications like determining molecular weight and oligomeric status are also summarized.
This document provides information on electrophoresis, including:
1. Electrophoresis is a technique used to separate charged particles like proteins and nucleic acids in an electric field based on their charge-to-mass ratio.
2. Agarose and polyacrylamide gels are commonly used supporting media that provide a matrix for particle separation.
3. The general procedure involves sample application, electrophoretic separation, staining to visualize bands, and detection/quantification of bands through densitometry.
Electrophoresis is a laboratory technique that separates biomolecules like DNA, RNA, and proteins based on their size and charge. It works by applying an electric current to move the charged molecules through a gel, with smaller molecules moving faster than larger ones. Common applications of electrophoresis include analyzing DNA and proteins from blood and urine samples. It allows researchers to examine molecules at high resolution and test things like antibiotic purity and vaccine concentrations.
This document discusses electrophoresis, which is the migration of charged particles through a liquid medium under the influence of an electric field. It defines key terms and describes the theory behind electrophoresis, factors that influence particle migration rates, and different electrophoresis techniques. Some main techniques covered are agarose gel electrophoresis, polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis. Troubleshooting tips for common issues are also provided.
Electrophoresis is a technique used to separate charged particles such as proteins, nucleic acids, and other molecules based on their size and charge. It works by applying an electric field to encourage the particles to migrate through a gel or other medium at different rates. The document discusses the components, principles, and applications of electrophoresis, including different types of gels, buffers used, and methods of visualization. It is commonly used in fields like molecular biology, genetics, and forensics.
This document provides an overview of electrophoresis. It discusses how electrophoresis works, involving the movement of charged particles through an electrolyte when subjected to an electric field. It then covers the history, factors affecting electrophoresis, types of electrophoresis including paper, SDS-PAGE, native gel, gradient gel, IEF gels, 2D gel electrophoresis, protein blotting, pulsed field gel electrophoresis, and capillary electrophoresis. It also discusses instrumentation, reagents, and applications of various electrophoresis techniques.
At any given pH, molecules exist as electrically charged species that will migrate toward the cathode or anode under the influence of an electric field, depending on their net charge. Electrophoresis techniques separate these charged particles using an electric current applied across a buffer solution and supporting medium like agarose gel or polyacrylamide. The basic equipment required consists of a power supply delivering current between electrodes in an electrophoresis unit, which can perform vertical or horizontal gel separations. Proteins and other analytes are visualized after separation by staining or other detection methods. Electrophoresis has numerous applications in fields like medicine, biochemistry, and food analysis.
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.
2. CONTENTS
INTRODUCTION
THEORY
TYPES OF ELECTROPHORESIS
MOVING BOUNDARY ELECTROPHORESIS
ZONE ELECTROPHORESIS
PRINCIPLE AND TYPES OF ZONE ELCTROPHORESIS
GEL ELECTROPHORESIS
PRINCIPLE OF GEL ELECTROPHORESIS
AGAROSE GEL PREPRATION AND INSTRUMENTATION
SAMPLE PREPRATION
GEL LOADING AND MICROPIPETTING TECHNIQUE
GEL STAINING
POLY ACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
PREPRATION OF PAG AND ITS COMPONENTS
COMPARISION OF FLATBED SYSTEM AND VERTICAL GEL
SYSTEM
ISO-ELECTRIC FOCUSSING
SDS-PAGE
4. ELECTROPHORESIS
Electrophoresis is also called Cataphoresis.
Electrophoresis is a separation technique in which ions
in colloidal solutions are separated based upon their
differences in size and charge when a high voltage is
applied to the solution[1]
Positive ions migrate to the negative electrode
(cathode), and negative ions migrate towards the positive
electrode (anode).
Was developed into workable system by Arne Tiselius of
Sweden in 1930. He won Nobel prize for the work in 1948.
Introduction:
5. Theory of electrophoresis
Mobility- rate of movement ,denoted by “μ”.
Particle to migrate in electric field requires a net electrostatic charge.
Consider a particle that is placed in a container of liquid, saturated
with the buffer and has a potential applied to it. The force “F” is equal
to:
F= QE Where,
Q= charge of the particle
E= field strength
As particle moves in the buffer it meets retardation force caused by
the viscosity of the solvent which can be expressed as:
Fs= 6πrημ Where,
fs = viscous retardation force
r = radius of particle in cm
η = viscosity of mediun in poise
μ = electrophoretic velocity in cm/sec
Now when both the forces are equal i.e
Fs = QE = 6πrημ
6. Hence mobility of particle can be defined as:
μ = υ/E where,
μ = mobility in cm2
volt-1
sec-1
E = volt cm-1
After substitution,
μ = Q/6πrη
Factor affecting the mobility[1]
Heat production
Smiling
Electro-osmosis correction
7. Moving boundary electrophoresis :
Density gradient electrophoresis
Iso-tachophoresis
Zone electrophoresis:
Paper electrophoresis
Gel electrophoresis
Capillary zone electrophoresis
Immuno- electrophoresis
Iso-electric focusing electrophoresis
Types of electrophoresis
8. Separation occurs due to difference
in mobility of molecules. Mobility is
proportional to m/e ratio.
The position of moving ions, which
forms a boundary, which is detected
by measuring the changes in
refractive index throughout solution.
The concentration gradients which
are formed during electrophoresis are
usually detected by optical method.
Moving boundary electrophoresis[2]
This method allows the charged species to migrate in a free moving
solution in absence of a supporting medium.
Samples are fractioned in a U shaped tube that has been filled with
unstabilized buffer.
An electrical field is applied by means of electrodes at the ends of
the tube.
Fig1: illustration of moving
boundary electrophoresis
9. Advantages:
1.Application to a wide variety of high molecular weight
substances.
Disadvantages [3]
1.Mixing of separated compound as a consequence of
thermal and density gradient as well as mechanical vibration.
2.Thermal vibration and mechanical vibration controlling is
difficult and expensive.
3.Detection of fraction separated with optical system also
adds to the expenses.
ADVANTAGE AND DISADVANTAGES OF MOVING
BOUNDARY ELECTROPHORESIS
10. ZONE ELECTROPHORESIS
Any electrophoretic technique in which components are
separated into zones or bands in a buffer and stabilized in
solid, porous, or any other support medium e.g.: paper strip,
agar gel or poly-acrylamide gel.
Separates macromolecular colloids e.g.. proteins in serum,
urine, Cerebrospinal fluids (CSF), erythrocytes; nucleic acids.
Types of zone electrophoresis are:-
1. Paper electrophoresis
2. Gel electrophoresis
3. Capillary zone electrophoresis
4. Immuno- electrophoresis
11. ADVANTAGES AND DISADVANTAGES OF ZONE
ELECTROPHORESIS
ADVANTAGES:
1. Useful in biochemical investigation.
2. Very small quantity of samples can be analyzed.
3. Useful to study both simple and complex mixtures equally.
4. Equipment cost is low and maintenance is easy.
5. Detection and visualization with various reagents and dyes
are possible
6. Permits 2-D electrophoresis for higher resolution.
7. Quantification by densitometry and auto-radiography can
be done
DISADVANTAGE:
1. Unsuitable for accurate mobility and iso-electric point
determination.
2. Complications such as capillary flow, electro osmosis,
adsorption and molecular sieving are introduced.
12. GEL ELECTROPHORESIS[4]
Gel electrophoresis is a widely used technique for the
analysis of nucleic acids and proteins. Gel electrophoresis is
routinely used for the preparation and analysis of DNA.
Gel electrophoresis is a procedure that separates molecules on the
basis of their rate of movement through a gel under the influence of an
electrical field.
Types of gel electrophoresis:-
1. One-dimensional - Agarose gel electrophoresis, Poly-
acrylamide gel electrophoresis (native or SDS-PAGE) and Iso-
electricfoccusing (IEF) or
2. Two dimensional - 2D-PAGE.
3. Capillary electrophoresis
13. +-
Power
DNA
PRINCIPLE OF GEL ELECTROPHORESIS
DNA is negatively charged
When placed in an electric field, DNA will migrate towards the
positive pole (anode).
An agarose gel is used to slow the movement of DNA and
separate by size.
14. Fig 2 :Scanning Electron Micrograph of Agarose Gel (1×1 µm)
• Polymerized agarose is porous, allowing for the movement of
DNA
15. +-
Power
DNA
How fast will the DNA migrate?
• Strength of the electrical field, buffer, density of agarose gel
• Size of the DNA
• Small DNA move faster than large DNA
• Gel electrophoresis separates DNA according to size
small
large
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
16. AGAROSE [5]
•Agarose is a linear polymer
extracted from seaweed.
• 0.7% - for large DNA (5-10
kb)
• 2%- for small DNA (0.2-
1kb)
• Recommended
concentration is 1%
•Agarose was first used in
biology when Robert Koch
used it as a culture medium
for Tuberculosis bacteria in
1882
Fig 3:- Agarose used for
electrophoresis
18. An agarose gel is prepared by
combining agarose powder and a
buffer solution.
Agarose
Buffer
Flask for boiling
Buffer used- Tris-borate buffer (pH
8.0) that contains EDTA (TBE);
EDTA is a chelating agent that
binds divalent cations such as
Mg++ that many nucleases
require for their activity; EDTA
thus protects the DNA from
enzymatic degradation[6]
Fig: 4
19. Casting tray
Gel combs
Power supply
Gel tank
Cover
Electrical leads
Electrophoresis Equipment
Fig 5: Electrophoresis Equipment
20. Gel casting tray & combs
Fig :6
Small 8x10 cm gels (minigels) are very popular.
The volume of agarose required for a minigel is around 30–50 mL,
for a larger gel it may be 250 mL.
21. Seal the edges of the casting tray and put in the combs. Place the casting
tray on a level surface. None of the gel combs should be touching the
surface of the casting tray.
Preparing the Casting Tray
Fig: 7
22. Fig 8:Agarose Fig: 9 Buffer Solution
Combine the agarose powder and buffer solution. Use a flask
that is several times larger than the volume of buffer.
23. Fig 10: Agarose is insoluble at
room temperature.
Gently swirl the solution periodically when heating to allow all the grains of
agarose to dissolve.
Be careful when boiling - the agarose solution may become superheated
and may boil violently if it has been heated too long in a microwave oven.
Melting the Agarose
Fig 11: The agarose solution is
boiled until clear.
24. Allow the agarose solution to cool slightly (~60ºC) and then
carefully pour the melted agarose solution into the casting tray.
Avoid air bubbles.
Pouring the gel
Fig :12
25. Each of the gel combs should be submerged in the melted agarose
solution.
Fig : 13
26. When cooled, the agarose polymerizes, forming a flexible gel. It
should appear lighter in color when completely cooled (30-45
minutes). Carefully remove the combs and tape.
Fig : 14
27. Place the gel in the electrophoresis chamber.
Fig: 15
28. buffer
Add enough electrophoresis buffer to cover the gel to a
depth of at least 1 mm. Make sure each well is filled with
buffer.
Cathode
(negative)
Anode
(positive)
wells
DNA
Fig: 16
29. 6X Loading Buffer:
• Bromophenol Blue (for color)
• Glycerol (for weight)
Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/
tracking dye). This allows the samples to be seen when loading
onto the gel, and increases the density of the samples, causing
them to sink into the gel wells.
Fig 17: prepared DNA sample
30. Loading the Gel
Carefully place the pipette tip over a well and gently expel the
sample. The sample should sink into the well. Be careful not to
puncture the gel with the pipette tip.
Fig 18
34. Place the cover on the electrophoresis chamber, connecting the
electrical leads. Connect the electrical leads to the power supply. Be
sure the leads are attached correctly - DNA migrates toward the
anode (red). When the power is turned on, bubbles should form on
the electrodes in the electrophoresis chamber.
The electrophoresis is run by 70-100 V/20-80 mA for about an hour or at
20 to 30 V overnight [6]
RUNNING THE GEL
Fig 22 Fig 23
36. 100
200
300
1,650
1,000
500
850
650
400
12,000 bp
5,000
2,000
DNA LADDER STANDARD
Inclusion of a DNA ladder (DNAs of know sizes) on the gel
makes it easy to determine the sizes of unknown DNAs.
-
+
DNA
migration
bromophenol blue
37. STAINING THE GEL
***CAUTION! Ethidium bromide is a powerful mutagen and is moderately
toxic [7]
• Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
• Ethidium bromide can be added to the gel and/or running
buffer before the gel is run or the gel can be stained after it has
run.
38. SAFER ALTERNATIVES TO ETHIDIUM BROMIDE
Methylene Blue
Carolina BLU Stain
EVA green [8]
Advantages
Inexpensive
Less toxic
No UV light required
No hazardous waste disposal
Disadvantages
Less sensitive
More DNA needed on gel
Longer staining/destaining time
stains Compounds
Amido black 10 B Proteins
Coomassie blue Proteins
Sudan black Lipid & lipoproteins
Ninhydrine Amino acid
Other stains which are used [1]
39. STAINING THE GEL
• Place the gel in the staining tray containing warm diluted stain.
• Allow the gel to stain for 25-30 minutes.
• To remove excess stain, allow the gel to destain in water.
• Replace water several times for efficient destain.
Fig 27
40. Ethidium Bromide requires an ultraviolet light source to visualize
DNA bands
under UV light in
an ethidium-
bromide-stained
gel is easily
visible if it
contains about
20 ng of DNA.
Fig 28
41. POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)
PAGE, is one of the most widely used electrophoresis techniques
and separates proteins through a polyacrylamide gel matrix[4]
Two types of PAGE can be carried out
1.Native PAGE- in which electrophoresis is carried out under non-
denaturing conditions and separation is based on the protein’s
charge and hydrodynamic size or
2. SDS PAGE - in which proteins are denatured prior to
electrophoresis and separation is based on a protein’s mass or
molecular weight.
The chemical SDS (sodium dodecyl sulphate) is an anionic
detergent which, in combination with DTT (dithiothreitol) or β-
mercaptoethanol, breaks intramolecular bonds in the protein
destroying any secondary, tertiary or quaternary structure.
This leaves only the linear primary amino acid structure of the
protein which will contain an overall negative charge proportional to
its mass, therefore allowing the proteins to be separated solely on
the basis of their molecular mass.
42. The gel typically consist of acrylamide, bisacrylamide, SDS, and a
buffer with an adjusted pH.
The solution may be degassed under a vacuum to prevent the
formation of air bubbles during polymerization.
A source of free radicals and a stabilizer such as ammonium
persulfate and TEMED are added to initiate polymerization.
The polymerization reaction results in a gel because of the added
bisacrylamide, generally about 1 part in 35 relative to acrylamide,
which can form cross-links between two polyacrylamide molecules.
The ratio of acrylamide to bisacrylamide can be varied for special
purposes.
The acrylamide concentration of the gel can also be varied,
generally in the range from 5% to 25%.
Lower percentage gels are better for resolving very high molecular
weight proteins, and viceversa.
PREPARING POLY-ACRYLAMIDE GELS[9]
43. COMPONENTS OF POLYACRYLAMIDE GEL ARE [9]
:-
1.Chemical buffer -to stabilizes the pH value to the desired value-
Tris , Bis-Tris or imidazole.
2.Counterion -to balance the intrinsic charge of the buffer ion and
also affect the electric field strength during electrophoresis – glycine
and tricine.
3.Acrylamide.
4.Bisacrylamide - used as cross linking agent .
5.Sodium Dodecyl Sulfate - denature native proteins
6.Ammonium persulfate - as an initiator for gel formation.
7.TEMED (N, N, N', N'-tetramethylethylenediamine)- stabilizes
free radicals and improves polymerization.
44. CHEMICALS FOR PROCESSING AND VISUALIZATION
Tracking dye:- Anionic dyes of a known electrophoresis mobility are
used like Bromophenol blue to follow colorless protein .
Being a highly mobile molecule it moves ahead of most proteins. As it
reaches the anodic end of the electrophoresis medium electrophoresis
is stopped.
Loading aids:- To ensure that the sample sinks to the bottom of the
gel glycerol and sucrose are used to increase the density of the
sample.
Coomassie Brilliant Blue R-250 (CBB) - is an anionic dye.
Proteins in the gel are fixed by acetic acid and simultaneously stained.
The excess dye incorporated into the gel can be removed by
distaining with the same solution without the dye. The proteins are
detected as blue bands on a clear background
46. ADVANTAGES OF PAGE ELECTROPHORESIS
1. Excellent separation on basis of size, shape, and charge
2. Pore size and the amount of cross linking can be controlled
3. Excellent resolution
4. Separation is rapid (30 min to a few hours)
5. Apparatus is relatively simple to operate
6. PAGE has a high loading capacity, up to 10 micrograms of
DNA can be loaded into a single well (1 cm x 1 mm) without
significant loss of resolution.
7. PAGE is an ideal gel system from which to isolate DNA
fragments for subcloning and other molecular biological
techniques.
47. As any other methods, PAGE also has disadvantages:
1. The mobility of the fragments can be affected by base
composition making accurate sizing of bands a problem.
2. Polyacrylamide quenches fluorescence, making bands
containing less than 25 ng difficult to visualize with
ethidium bromide staining.
3. Quantification difficult by itself - Increased when combined
with procedures using radiolabeled or photometric
markers
4. Acrylamide is a neurotoxin
48. Flatbed Systems: Vertical Systems:
Gel thickness is limited, because
cooling is only possible from one side
Higher protein loading capacity,
because thicker gels can be used,
which are cooled from both sides
Blotting is easier because of higher
gel thickness
One gel per instrument is run Multiple gel runs possible
Very versatile for different methods,
ideal for isoelectric focusing
Limited technical possibilities, not
optimal for isoelectric focusing
Thin layers can easily be used,
easy sample application
The thinner the gel, the more
complicated is sample application
Easy to handle and to clean,
no glass plates necessary, thus ideal
for routine applications
Many pieces to set up and to clean
COMPARISON OF FLATBED AND VERTICAL GEL SYSTEMS[4]
49. ISO ELECTRIC FOCUSSING[1] [4]
It is mainly used for a separation of electrolytes such as proteins.
When electrophoresis is run in solution buffered at constant pH,
proteins having net charge will migrate towards opposite electrode.
The use of pH gradient across supporting medium causes each protein
to migrate to an area of specific pH.
A sharp well defined protein bands occur at the point where iso electric
point equals to pH of gradient
Separation is carried out on gels on which a stable a pH gradient has
been established.
An ampholytic compound has a pH at which it is neutral.
The pH gradient is achieved by impregnating the gel with polyamino-
polycarboxylic acids.
When subjected to electric field these migrate and come to rest in
order of their pH.
Thus each ampholyte migrates in applied field until it reaches a
position on the plate where pH of medium is equal to iso electric point.
50. At this point ampholyte is in its zwitter ion form and is
neutral.
Thus it losses electrophoretic mobility and becomes focused
in narrow zone at this point.
Fig 31: illustrating IEF
51. Advantages:
1.Spreading of bands is minimized.
2.Proteins that differ by little as 0.01pH can be adequately resolved
Disadvantages:
1.As carrier ampholytes are used in high concentration, a high voltage
power supply is necessary. As a result the electrophoretsis may be
affected.
ADVANTAGES AND DISADVANTAGES OF IEF
52. SDS-PAGE (PolyAcrylamide Gel Electrophoresis)[9]
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis, is a technique widely used in biochemistry, forensics,
genetics and molecular biology:
1.To separate proteins according to their electrophoretic mobility (a
function of length of polypeptide chain or molecular weight).
2.To separate proteins according to their size, and no other physical
feature.
SDS (sodium dodecyl sulfate) is a detergent (soap) that can
dissolve hydrophobic molecules but also has a negative charge
(sulfATE) attached to it.
SDS (the detergent soap) breaks up hydrophobic areas and coats
proteins with negative charges thus overwhelming positive charges in
the protein.
The detergent binds to hydrophobic regions in a constant ratio of
about 1.4 g of SDS per gram of protein.
Therefore, if a cell is incubated with SDS, the membranes will be
dissolved, all the proteins will be solublised by the detergent and all
the proteins will be covered with many negative charges
53. Fig 32 : illustrating the effect of SDS over protein moleculesfi
54. COMPONENTS OF SDS PAGE GEL[9]
Running Buffer: Tris/Glycine:
Glycine(pKa=9.69) is a trailing
ion (or slow ion). In other
words it runs through the gel
slower then the slowest protein
at a pH above 8.0.
Stacking Gel: Is prepared w/Tris/HCL buffer pH 6.8, ~2pH units
lower than running buffer. Large pore polyacrylamide used to align
and create a thin starting zone of the protein of apx. 19µm on top of
the resolving gel.
Resolving Gel: Small pore polyacrylamide gel (3 - 30% acrylamide
monomer) typically made using a pH 8.8 Tris/HCl buffer. Resolves
protein ~24 – 205 kDa
Fig: 32
55. TWO DIMENSIONAL ELECTROPHORESIS
Separation of hundreds of proteins
based on:
Isoelectric point
Molecular weight
Popular method for protein display
and proteomics-one spot at a time
Permits simultaneous detection,
display, purification, identification,
quantification, pI, and MW.
Robust, reproducible, simple, cost
effective, scalable
Provides differential quantification
using Differential 2D Gel
Electrophoresis (DIGE)
Fig33: The principle of the 2-D electrophoresis according to
O’Farrell (1975).
56. PROCESSES INVOLVED IN 2D GEL ELECTROPHORESIS
Protein isolation and quantification
Isoelectric focusing (first dimension)
SDS-PAGE (second dimension)
Visualization of proteins spots with Dye
Identification of protein spots with Mass Spec
57. CAPILLARY ELECTROPHORESIS[1]
Also known as:
High performance capillary electrophoresis
Capillary zone electrophoresis
Free solution capillary electrophoresis
Capillary electrophoresis is a micro-electrophoretic system in
which separation takes place in a 10-100μm internal diameter,
fused quartz, hollow capillary tube from 30-100 cm long with
each end immersed in a buffer.
DC applied upto 300V/cm.
WHY CAPILLARY ELECTROPHORESIS?
Reduces the problem resulting from heating effect- more
surface area to volume ratio hence more heat dessipation.
Reduce zone broadening.
58. Forces associated with the capillary electrophoresis are
Electro-osmotic flow (EOF)
Electro-phoretic separation
Under these influence all components in the sample travels in
one direction towards cathode.
The electro-osmotic flow (EOF) is caused by applying high-
voltage to an electrolyte-filled capillary.
This flow occurs when the buffer running through the silica
capillary has a pH greater than 3 and the SiOH groups lose a
proton to become SiO-
ions.
The capillary wall then has a negative charge, which develops
a double layer of cations attracted to it.
The inner cation layer is stationary, while the outer layer is
free to move along the capillary.
The applied electric field causes the free cations to move
toward the cathode creating a powerful bulk flow.
in CE potential applied is greater then normal electrophoresis
i.e 300V/cm this facilitates electro-phoretic separation.
PRINCIPLE OF CAPILLARY ELECTROPHORESIS
60. COMPONENTS OF CE
1. COLUMN
2. SAMPLE
3. BUFFERS
4. MODIFIERS
5. POWER SUPPLY
6. DETECTORS
COLUMNS:- borosilicate or fused quartz
Dimensions : internal diameter 10-100μm
length 30-100cm
capillary wall 300-600 μm
SAMPLE:- 5-50nL
Added by placing one end of the capillary into sample
containers raised to a set height (10cm) for a measured
time (30sec) and allowing a siphoning action takes place
or place one end into the sample and apply a potential for
a short time.
61. BUFFERS: 0.5mM concentration is common
10mM mannitol
0.05M pH 7 phosphate, borate etc
MODIFIERS:- used if EOF is not demanded.
these alter the direction and rate of EOF
reverse flow- cetyltrimethyammoniunbromide
tetradecyltrimethylammonium bromide
zero flow-s-benzylthiouronium chloride
reduce flow- methanol
increase flow- acetonitrile
POWER SUPPLY:- faster heat dessipation hence high
potential of 300V/cm can be applied.
DETECTORS:- U-V-visible detectors, fluorescence
detectors, mass spectrometry,radioisotopes,
conductometry and amperometry etc
62. 1. CE has a flat flow, compared to the pumped parabolic flow of the
HPLC. The flat flow results in narrower peaks and better resolution.
2. CE has a greater peak capacity when compared to HPLC—CE
uses millions of theoretical plates.
3. HPLC is more thoroughly developed and has many mobile and
stationary phases that can be implemented.
4. HPLC has more complex instrumentation, while CE is simpler for
the operator.
5. HPLC has such a wide variety of column lengths and packing,
whereas CE is limited to thin capillaries.
6. CE require less sample volume then HPLC.
Capillary Electrophoresis versus High Performance Liquid
Chromatography (HPLC) [10]
CE HPLC
Length of column 30-100cm 3-25 cm,25-
50cm(microbore)
Internal diameter 10-100μm 4-6 mm,1-2 mm(microbore
Sample volume 5-50 nL 20μL
63. COMPARISION OF CLASSICAL GEL AND CE
Classical gel electrophoresis Capillary electrophoresis
Gels – polyacrylamide or agarose,
Slabs length and width 5-25 cm
Columns length- 7-10cm,internal
diameter- 5mm
Fused quartz capillary
Length- 30-100cm, internal diameter-10-
100μm
Electrophoretic separation Electrophoretic and electro-osmotic
Applied field100-2kV Applied field 10-50 kV
Heat dessipation is slow from column
and quicker in slabs
Heat dessipation is rapid
0.05-0.5M electrolyte for conductivity
and stability
Sample volume 1-50 μL
Different sample can be analysed
Same as classical gel
Sample volume 1-50nL
Only one sample analysed at a time
Chromogenic agent or staining agent for
detection of solute
HPLC detectors ,U-V absorbance and
fluorescence are commonly used
Slow, limited resolution and time
consuming.
High resolution, efficiency and
sensitivity, is modrately fast
64. ADVANTAGES AND DISADVANTAGES OF CE
Advantages
Offers new selectivity, an alternative to HPLC
Easy and predictable selectivity
High separation efficiency (105
to 106
theoretical plates)
Small sample sizes (1-10 ul)
Fast separations (1 to 45 min)
Can be automated
Disadvantages
Cannot do preparative scale separations
Reproducibility problems
65. REFRENCES
1. Milons
2. Elctrophoresis in practice, fourth edition Reiver Westermeir
3. Instrumental method of chemical analysis. By B.K.Sharma, page
no- 269
4. Randox Research & Development catalogue 2009/10
5. HHMI undergraduate research studio – Freshman, Biology
section, Fall 2007 – Agarose Gel Electrophoresis.
6. www.wikipedia.org/wiki/Gel_Electrophoresis.
7. www.wikipedia.org/wiki/ethidium_bromide
8. Biotech.about.com/../DNA strain.htm
9. www.wikipedia.org/wiki/SDS_PAGE
10. http://elchem.kaistac.kr/chem-ed