Sds page gel electrophoresis
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This document discusses SDS-PAGE gel electrophoresis, which is used to separate proteins by size. It describes how polyacrylamide gels are formed through chemical polymerization and how proteins are denatured and given a uniform charge by boiling them with SDS and 2-mercaptoethanol. When a voltage is applied, SDS-coated protein complexes migrate through the stacking and resolving gels at rates dependent on their molecular weights. The positions of separated proteins can then be visualized by staining the gel.
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SDS PAGE
SDS-PAGE is a technique used to separate proteins by molecular weight. Proteins are denatured and given a negative charge by SDS detergent before running through a polyacrylamide gel matrix by electrophoresis. Smaller proteins migrate faster through the gel, allowing separation by size. After electrophoresis, proteins bands can be visualized using stains like Coomassie blue or silver stain to analyze characteristics like molecular weight, purity, and subunit composition.
Agarose gel electrophoresis
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Sds page
Polyacrylamide gel electrophoresis (PAGE) is a method used to separate macromolecules like proteins or nucleic acids based on their size and charge. It works by applying an electric current to move the charged molecules through a polyacrylamide gel matrix. Smaller molecules move faster through the gel's pores than larger ones. SDS-PAGE specifically uses sodium dodecyl sulfate detergent to denature proteins and impart a uniform negative charge, allowing separation based primarily on size. The document discusses the principles, procedures, applications and different types of PAGE in detail.
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Agrose gel electrophoresis
This document summarizes the process of agarose gel electrophoresis. Agarose gel is prepared by combining agarose powder with a buffer solution to establish pH and conductivity. Samples are loaded into wells in the gel and an electric current is applied, causing DNA fragments to migrate through the gel at rates corresponding to their size. Agarose gel electrophoresis is used to separate and analyze DNA fragments for applications such as estimating DNA size, analyzing polymerase chain reaction products, and extracting DNA fragments for further purification.
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SDS-PAGE is a technique used to separate proteins by molecular weight. Proteins are denatured and given a negative charge by SDS detergent before running through a polyacrylamide gel matrix by electrophoresis. Smaller proteins migrate faster through the gel, allowing separation by size. After electrophoresis, proteins bands can be visualized using stains like Coomassie blue or silver stain to analyze characteristics like molecular weight, purity, and subunit composition.
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Sds page
Polyacrylamide gel electrophoresis (PAGE) is a method used to separate macromolecules like proteins or nucleic acids based on their size and charge. It works by applying an electric current to move the charged molecules through a polyacrylamide gel matrix. Smaller molecules move faster through the gel's pores than larger ones. SDS-PAGE specifically uses sodium dodecyl sulfate detergent to denature proteins and impart a uniform negative charge, allowing separation based primarily on size. The document discusses the principles, procedures, applications and different types of PAGE in detail.
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Agrose gel electrophoresis
This document summarizes the process of agarose gel electrophoresis. Agarose gel is prepared by combining agarose powder with a buffer solution to establish pH and conductivity. Samples are loaded into wells in the gel and an electric current is applied, causing DNA fragments to migrate through the gel at rates corresponding to their size. Agarose gel electrophoresis is used to separate and analyze DNA fragments for applications such as estimating DNA size, analyzing polymerase chain reaction products, and extracting DNA fragments for further purification.
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SDS- Polyacrylamide Gel Electrophoresis
SDS-Polyacrylamide Gel Electrophoresis
What is SDS?
Preparation of Gel
Process of SDS-PAGE
Visualization of protein bands
SDS-PAGE is differentiated into two systems.
*continuous sds-page
*discontinuous sds-page.
Polyacrylamide is used to form a gel, a matrix of a pores which allow the molecules migrate at different rates.
Negatively charged detergent sodium dodecyl sulfate.
Used to denature and linearize the proteins
Coated the proteins with negatively charged.
SDS-page is a technique that used to separate proteins according to their molecular size through the gel.
Proteins are unfolded and migrate from cathode to anode terminal at different rates.
Molecular weight is determined by compare the result with a standard curve of relative motility of standard proteins.
Visualizes the band under UV light.
Types of stains;
Coomassie Blue;
* Coomassie Brilliant Blue staining The Coomassie dyes R-250 and G-250 bind to proteins stoichiometrically through their sulfonic acid groups.
* . The interactions between dye and protein are Van der Waals and ionic. The sulfonic acid groups interact with positive amine groups. Therefore coomassie dye binds to wide range of proteins.
* Limited to ~100ng of protein.
Silver stain;
*most sensitive test
*detection limit 0.1-1.0ng of protein
The size of pores is determined by the concentration of acrylamide.
The higher the concentration, the smaller the size of pores.
Discontinuos sds-page consist of two different gels.
*stacking gel -4%of acrylamide
*separating gel-range from 5-15% of acrylamide.
Hydrophobic interaction chromatography (1)
Hydrophobic interaction chromatography (HIC) is used to purify biomolecules like proteins by exploiting differences in hydrophobicity. It works by separating substances based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix. HIC is useful for downstream purification as it can handle large sample volumes with high salt concentrations and is gentler than other methods, maintaining protein activity. Factors like ligand type, salt concentration, pH, and temperature affect HIC separation. The document evaluates HIC for monitoring plasmid DNA purification, finding it is a simple, rapid and reproducible method to assess purity.
SDS PAGE
This document describes SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), a technique used to separate proteins by their molecular weight. SDS denatures proteins and gives them a uniform charge. This allows proteins to be separated solely by size as they migrate through a polyacrylamide gel under an electric field. Smaller proteins migrate faster. The document outlines the SDS-PAGE method and preparation of samples, gels, buffers and staining for visualization and analysis of separated proteins. Applications include determining protein purity, molecular weight and peptide mapping.
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widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
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Poly acrylamide gel electrophoresis (page)
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.
PAGE- Electrophoresis
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
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Gel filtration chromatography
Gel-filtration chromatography, also known as size-exclusion chromatography, separates molecules based on their size. Larger molecules pass through the porous gel beads quickly while smaller molecules diffuse into the pores and are delayed. This allows the separation of molecules like proteins, nucleic acids, and polymers by molecular weight. It is a gentle technique that can be used to purify biomolecules without altering buffer conditions.
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This presentation includes the principle involved, chemistry, procedure, and application of various advance molecular biology like SDS PAGE, Western Blotting, and ELISA.
SDS PAGE is widely used to analyze the proteins in complex extracts.
The polyacrylamide gels are used to separate proteins.
Polyacrylamide is inert, and hence, shows no interaction with the protein being separated and forms a matrix.
Size of the pores in the gel can be controlled by adjusting the concentration of acrylamide.
Acrylamide undergoes polymerization in order to form a gel. Hence, APS (ammonium per sulphate) & TEMED (N,N,N’,N’-tetramethylethylenediamine) are added to initiate the process of polymerization.
It's application includes separation of protein mixture on separating gel and their identification using different techniques like western blotting.
Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells.
Western blots are effective in detecting low nanogram to low picogram amounts of target protein, depending on the antibodies used and the detection substrate chosen. If the target is suspected to be of very low abundance, or if there is no detectible signal on the blot, then it may be necessary to concentrate, immunoprecipitate, or fractionate the starting material.
This technique is used to study cell signalling pathways, cell cycle pathways, drug action pathways, protein-protein interaction.
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones.
In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.
Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product.
The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
ELISA begins with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate.
This is followed by a blocking step in which all unbound sites are coated with a blocking agent.
Following a series of washes, the plate is incubated with enzyme-conjugated antibody.
Another series of washes removes all unbound antibody.
A substrate is then added, producing a calorimetric signal. Finally, the plate is read.
It's types include Direct ELISA, Indirect ELISA, Sandwich ELISA and competitive ELISA. This technique is used to determine serum antibody concentrations, potential food allergens (milk, peanuts, almonds), detection of antigens and antibodies, disease outbreaks.
Final - SDS-PAGE electrophoresis.docx
SDS-PAGE is a technique used to separate proteins according to their electrophoretic mobility. It involves treating proteins with SDS detergent and running them on a polyacrylamide gel with an electric current, allowing smaller proteins to migrate faster. The document provides details on the history, principles, required materials, procedure, applications, and advantages/disadvantages of SDS-PAGE. It is commonly used to determine protein molecular weights, compare protein compositions, and analyze purity.
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This document describes Fast Protein Liquid Chromatography (FPLC), which is a chromatography system used to separate proteins and other biomolecules. FPLC uses stationary phases like resins and mobile phases like buffers. It works on the principles of size exclusion, ion exchange, and affinity chromatography. The key components of an FPLC system include pumps, mixers, injection valves, columns, fraction collectors, and detectors. Compared to HPLC, FPLC uses lower pressures, inert materials, and larger columns. Its advantages include reproducibility, simple operation, and suitability for analytical and preparative purposes. Common applications of FPLC include protein analysis, purification of venoms and plasma proteins.
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Agarose gel electrophoresis is a method to separate DNA fragments by size using an agarose gel matrix and electric current. Shorter DNA fragments migrate faster and farther than longer ones. DNA is visualized by staining with ethidium bromide and viewing under UV light. Agarose concentration determines resolution, with 0.8% gels best for separating large 5-10kb fragments and 2% for small 0.2-1kb fragments. Applications include estimating DNA size, analyzing PCR products, and separating DNA for further analysis.Agarose Gel Electrophoresis
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Cellulose acetate electrophoresis
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SDS-Polyacrylamide Gel Electrophoresis
What is SDS?
Preparation of Gel
Process of SDS-PAGE
Visualization of protein bands
SDS-PAGE is differentiated into two systems.
*continuous sds-page
*discontinuous sds-page.
Polyacrylamide is used to form a gel, a matrix of a pores which allow the molecules migrate at different rates.
Negatively charged detergent sodium dodecyl sulfate.
Used to denature and linearize the proteins
Coated the proteins with negatively charged.
SDS-page is a technique that used to separate proteins according to their molecular size through the gel.
Proteins are unfolded and migrate from cathode to anode terminal at different rates.
Molecular weight is determined by compare the result with a standard curve of relative motility of standard proteins.
Visualizes the band under UV light.
Types of stains;
Coomassie Blue;
* Coomassie Brilliant Blue staining The Coomassie dyes R-250 and G-250 bind to proteins stoichiometrically through their sulfonic acid groups.
* . The interactions between dye and protein are Van der Waals and ionic. The sulfonic acid groups interact with positive amine groups. Therefore coomassie dye binds to wide range of proteins.
* Limited to ~100ng of protein.
Silver stain;
*most sensitive test
*detection limit 0.1-1.0ng of protein
The size of pores is determined by the concentration of acrylamide.
The higher the concentration, the smaller the size of pores.
Discontinuos sds-page consist of two different gels.
*stacking gel -4%of acrylamide
*separating gel-range from 5-15% of acrylamide.
Hydrophobic interaction chromatography (1)
Hydrophobic interaction chromatography (HIC) is used to purify biomolecules like proteins by exploiting differences in hydrophobicity. It works by separating substances based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix. HIC is useful for downstream purification as it can handle large sample volumes with high salt concentrations and is gentler than other methods, maintaining protein activity. Factors like ligand type, salt concentration, pH, and temperature affect HIC separation. The document evaluates HIC for monitoring plasmid DNA purification, finding it is a simple, rapid and reproducible method to assess purity.
SDS PAGE
This document describes SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), a technique used to separate proteins by their molecular weight. SDS denatures proteins and gives them a uniform charge. This allows proteins to be separated solely by size as they migrate through a polyacrylamide gel under an electric field. Smaller proteins migrate faster. The document outlines the SDS-PAGE method and preparation of samples, gels, buffers and staining for visualization and analysis of separated proteins. Applications include determining protein purity, molecular weight and peptide mapping.
Polyacrylamide gel electrophoresis
PAGE is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels use as the support matrix.
widely used and has very much importance.
COMPLETE PROCEDURE & USES are described in the slide.
differential centrifugation
Differential centrifugation is a technique used to separate cell organelles based on their densities. It involves homogenizing tissue to break open cells and mix organelle contents. The homogenate is then centrifuged at increasing speeds, causing organelles like mitochondria and lysosomes to pellet out after centrifuging at 1000g for 15 minutes. Repeating this process with the supernatant at higher speeds allows separation of organelles into fractions based on their sedimentation rates in a centrifugal field. While convenient and economical, differential centrifugation yields impure preparations and poor recovery of organelles.
Poly acrylamide gel electrophoresis (page)
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.
PAGE- Electrophoresis
It is a subtype of the gel electrophoresis whereby the normal gel is replaced with polyacrylamide gels used as support media.
Gels are made by free radical-induced polymerization of acrylamide and N,N’-Methylenebisacrylamide.
It is the most widely used technique of electrophoresis.
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This document discusses SDS-PAGE (sodium dodecyl sulphate- polyacrylamide gel electrophoresis), the most widely used method for analyzing protein mixtures. SDS-PAGE separates proteins based on their size. The sample is treated with SDS and beta-mercaptoethanol to denature and negatively charge the proteins. Proteins then migrate through a stacking gel and separating gel based on their charge and size. SDS-PAGE is useful for protein purification, determining molecular weight, and identifying disulfide bonds.
Gel filtration chromatography
Gel-filtration chromatography, also known as size-exclusion chromatography, separates molecules based on their size. Larger molecules pass through the porous gel beads quickly while smaller molecules diffuse into the pores and are delayed. This allows the separation of molecules like proteins, nucleic acids, and polymers by molecular weight. It is a gentle technique that can be used to purify biomolecules without altering buffer conditions.
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This document discusses agarose gel electrophoresis, which is used to separate DNA fragments by size. It begins by explaining the basic principle, which is that charged DNA fragments will move through an agarose gel under the influence of an electric field, with smaller fragments moving faster. It then describes how agarose gel is prepared by mixing agarose with buffer and casting it. Different types of gels, like agarose and polyacrylamide, can be used depending on the size of DNA fragments. The document outlines the steps for running samples on a gel, detecting DNA bands with ethidium bromide staining, and some applications like capillary and pulsed-field gel electrophoresis.
Flow cytometry and fluorescence activated cell sorting (FACS)
Flow cytometry is a technology that analyzes physical and chemical characteristics of particles in fluid as they pass through a laser. It is used for cell counting, sorting, biomarker detection, and protein engineering. The basic principle is passing cells in single file past a laser for detection, counting, and sorting. It has applications in leukocyte analysis, DNA analysis, detecting enzymatic deficiencies, minimal residual disease, detecting autoantibodies, fetal-maternal hemorrhage quantification, and reticulocyte analysis.
Electrophoretic mobility shift assay
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
2 -D GEL ELECTROPHORESIS
wo-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first independently introduced by O'Farrell and Klose in 1975.
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Similar to Sds page gel electrophoresis
SDS PAGE, WESTERN BLOTTING, AND ELISA
This presentation includes the principle involved, chemistry, procedure, and application of various advance molecular biology like SDS PAGE, Western Blotting, and ELISA.
SDS PAGE is widely used to analyze the proteins in complex extracts.
The polyacrylamide gels are used to separate proteins.
Polyacrylamide is inert, and hence, shows no interaction with the protein being separated and forms a matrix.
Size of the pores in the gel can be controlled by adjusting the concentration of acrylamide.
Acrylamide undergoes polymerization in order to form a gel. Hence, APS (ammonium per sulphate) & TEMED (N,N,N’,N’-tetramethylethylenediamine) are added to initiate the process of polymerization.
It's application includes separation of protein mixture on separating gel and their identification using different techniques like western blotting.
Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells.
Western blots are effective in detecting low nanogram to low picogram amounts of target protein, depending on the antibodies used and the detection substrate chosen. If the target is suspected to be of very low abundance, or if there is no detectible signal on the blot, then it may be necessary to concentrate, immunoprecipitate, or fractionate the starting material.
This technique is used to study cell signalling pathways, cell cycle pathways, drug action pathways, protein-protein interaction.
ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones.
In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.
Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product.
The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.
ELISA begins with a coating step, in which the first layer, consisting of a target antigen or antibody, is adsorbed onto a 96-well polystyrene plate.
This is followed by a blocking step in which all unbound sites are coated with a blocking agent.
Following a series of washes, the plate is incubated with enzyme-conjugated antibody.
Another series of washes removes all unbound antibody.
A substrate is then added, producing a calorimetric signal. Finally, the plate is read.
It's types include Direct ELISA, Indirect ELISA, Sandwich ELISA and competitive ELISA. This technique is used to determine serum antibody concentrations, potential food allergens (milk, peanuts, almonds), detection of antigens and antibodies, disease outbreaks.
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SDS-PAGE is a technique used to separate proteins according to their electrophoretic mobility. It involves treating proteins with SDS detergent and running them on a polyacrylamide gel with an electric current, allowing smaller proteins to migrate faster. The document provides details on the history, principles, required materials, procedure, applications, and advantages/disadvantages of SDS-PAGE. It is commonly used to determine protein molecular weights, compare protein compositions, and analyze purity.
SDS-PAGE Electrophoresis.pptx
SDS PAGE, or sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used to separate proteins by molecular weight. Proteins are denatured and given a negative charge by SDS, allowing them to be separated by size as they migrate through a polyacrylamide gel under an electric field. Key steps include sample preparation with SDS and reducing agents, casting the polyacrylamide gel, running electrophoresis to separate proteins by size, and staining to visualize the separated protein bands.
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This presentation consist of all information regarding sodium dodecyl sulphate PAGE. The general information regarding electrophoresis and its main types are also included in this presentation.
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Sodium dodecyl sulphate - Polyacrylamide Gel Electrophoresis (SDS - PAGE) is a technique used for the separation of deoxyribonucleic acid (DNA) ,Ribonucleic acid (RNA) And protein molecules according to their size and electrical charge.
Electrophoresis & its types
Electrophoresis is a technique used to separate charged particles such as proteins or nucleic acids. It involves applying an electric field to migrate these particles through a buffer or gel based on their size and charge. There are several types of electrophoresis including paper, gel, capillary, and moving boundary which utilize different supporting media and techniques to achieve high resolution separations. Electrophoresis is widely used in biochemistry and molecular biology for analytical purposes.
electrophoresis
Electrophoresis is a technique used to separate charged molecules like proteins and nucleic acids. It works by applying an electric field to migrate these molecules through a buffer or gel based on their size and charge. This document discusses different electrophoresis techniques including paper, gel, capillary electrophoresis and isoelectric focusing. It provides details on how each technique works, advantages and applications.
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Buffers resist changes in pH and consist of a conjugate acid-base pair where the ratio of proton acceptor to proton donor is near unity. A webinar discusses how biological buffers can impact reproducibility and explores considerations for buffer selection and use to improve reproducibility. Key factors include how buffers may interact with metals and biological materials and tips for proper buffer selection and use. [END SUMMARY]
ELECTRPOPHOROSIS
1. Electrophoresis is a physical method of analysis that separates compounds based on their ability to acquire an electric charge. Charged particles migrate through a conducting medium under the influence of an external electric field.
2. There are different types of electrophoresis including zone electrophoresis using paper, gels, or thin layers, and moving boundary electrophoresis including capillary and isotachophoresis.
3. Gel electrophoresis uses a gel like agarose or polyacrylamide as the supporting medium, allowing separation based on size and charge. SDS-PAGE separates proteins based on molecular weight by coating proteins with SDS.
Electrophoresis
This document provides information on electrophoresis techniques. It discusses how electrophoresis separates charged molecules like proteins and nucleic acids using an electric current. The key techniques covered are:
1. SDS-PAGE, which uses sodium dodecyl sulfate to denature proteins and give them a uniform negative charge for separation by size in a polyacrylamide gel.
2. Native PAGE, which separates intact proteins by their charge-to-size ratio.
3. Isoelectric focusing, which separates proteins based on their isoelectric point in a pH gradient gel.
It also discusses two-dimensional electrophoresis, which combines isoelectric focusing and SDS-PAGE to better resolve complex protein mixtures. The document
advanced electrophoresis
Electrophoresis is a technique used to separate molecules based on their charge and size. It works by applying an electric field to move charged molecules through a medium like agarose gel or polyacrylamide gel at different rates. Key factors that determine a molecule's movement include its net charge, size, shape, and the strength of the electric field. Variations of electrophoresis, like isoelectric focusing and two-dimensional electrophoresis, allow for highly precise separations of proteins and other biomolecules. Emerging techniques like pulsed field gel electrophoresis and capillary electrophoresis provide even higher resolution.
Electrophoresis
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
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.
Purification techniques
Protein purification techniques can be categorized into those based on molecular size, solubility, and electric charge. Size-based techniques include dialysis, ultrafiltration, and size-exclusion chromatography which separate proteins based on their ability to pass through semi-permeable membranes or porous beads. Solubility-based techniques include isoelectric precipitation and salting out which alter a protein's solubility by adjusting pH or salt concentration. Charge-based techniques such as ion-exchange and electrophoresis separate proteins using their net electric charge in an applied electric field or ion-exchange column.
1D PAGE (Poly Acrylamide Gel Electrophoresis)
This document provides an overview of one-dimensional polyacrylamide gel electrophoresis (1D-PAGE), which separates biological macromolecules like proteins and nucleic acids based on their electrophoretic mobility. It describes how charged molecules migrate in an electric field, the basic equipment used, and factors that affect separation quality like heat dissipation. It also discusses different gel compositions and concentrations used depending on the size of molecules separated, and variants of 1D-PAGE like SDS-PAGE which separates proteins by size.
Global analysis of protein by kk sahu sir
INTRODUCTION
GEL ELECTROPHORESIS
PRINCIPLE
BASIC CONCEPT
SUPPORT MEDIA
BUFFERS
APPARATUSS
AGE
PAGE
SDSPAGE
APPLICATIONS
CONCLUSIONS
BIBLIOGRAPHY
Electrophoresis and various techniques
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.
PAGE(Polyacrylamide gel electrophoresis).pptx
Here I have Explained About Polyacrylamide Gel Electrophoresis . What is The Principle of Electrophoresis? What is Gel Electrophoresis? Types of Gel Electrophoresis. What is PAGE?
How PAGE Run?
Why SDS PAGE?
mpr-SDS PAGE and other advanced techniques.pptx
Biotech
M pharma 2nd send'
dr harisingh gour sagar university
MUGDHA's SEMINAR MSc sem 1.pptx
Gel electrophoresis is a technique used to separate biomolecules like DNA, RNA, and proteins based on their size and charge. It works by applying an electric current to move the molecules through a gel, where smaller molecules move faster than larger ones. The document discusses the different types of gels used like agarose, polyacrylamide, and starch gels. It also explains the basic process which involves loading samples into wells, applying a current, and staining bands to visualize the separated biomolecules. Key applications are determining molecular weights and separating DNA or protein fragments.
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Sds page gel electrophoresis
- 1. SDS PAGE GEL ELECTROPHORESIS Ajay Prakash Uniyal , Central University Of Punjab
- 2. General Introduction SDS-PAGE is widely used to analyze the proteins in complex extracts. The system actually consists of two gels - a resolving (aka running) gel in which proteins are resolved on the basis of their molecular weights (MWs) and a stacking gel in which proteins are concentrated prior to entering the resolving gel. Gel matrices are permeated with networks of pores through which the molecules move. The amount of resistance that the matrix presents to the movement of a molecule depends on the diameter of the pore as well as the size and geometry of the molecule
- 3. Chemistry of acrylamide polymerization • The polyacrylamide gels used to separate proteins are formed by the chemical polymerization of acrylamide and a cross- linking reagent, N,N’methylenebisacrylamide. • Polymerization occurs because of free oxygen radicals that react with the vinyl groups in acrylamide and bisacrylamide. • The oxygen radicals are generated from the catalyst, ammonium persulfate (APS), when it reacts with a second catalyst, N,N,N’,N’-tetramethylethylenediamine (TEMED).
- 5. Proteins are mixtures of hydrophobic and hydrophilic amino acids and that the primary sequence of the protein determines its final folded form. To resolve the proteins in a sample according to their size, investigators must convert the proteins to a uniform geometry and impart a uniform charge/mass ratio to the proteins. In SDSPAGE, the solution is to denature the proteins by boiling them with the anionic detergent, sodium dodecyl sulfate (SDS) and 2-mercaptoethanol. The combination of heat and detergent is sufficient to break the many noncovalent bonds that stabilize protein folds, and 2- mercaptoethanol breaks any covalent bonds between cysteine residues.
- 6. SDS is an amphipathic molecule, consisting of a hydrophobic 12- carbon chain and a hydrophilic sulfate group. The SDS hydrocarbon chain permeates the protein interior and binds to hydrophobic groups, reducing theprotein to a random coil, coated with negatively charged detergent molecules all along its length. Denatured proteins bind quite a lot of SDS, amounting to ~1.4 g SDS/g protein, or ~one SDS molecule for every two amino acids. Once a voltage is applied, the chloride ions in the sample buffer and stacking gel move rapidly toward the positive pole, forming the leading edge of a moving ion front. Glycine molecules have very little charge in the stacking gel, so they migrate at the rear of the moving ion front. This difference in chloride and glycine mobility sets up a steep voltage gradient in the stacking gel that sweeps along the negatively charged protein-SDS complexes
- 7. • Dramatic changes occur as the glycine ions enter the running gel. The pH of the running gel is closer to the pKa of the glycine amino groups, so a significant fraction of the glycine molecules assume a negative charge. Negatively charged glycine molecules begin to move at the same rate as the chloride ions, thereby eliminating the voltage difference that controlled protein mobility through the stacking gel. The pores in the running gel are much smaller than those of the stacking gel, so the pores present frictional resistance to the migration of proteins • The sample buffer used for SDS-PAGE contains a tracking dye, bromophenol blue (BPB), which will migrate with the leading edge of the proteins being separated on the gel.
- 8. • To visualize the positions of proteins after electrophoresis is complete, investigators stain the gels with various dyes that bind noncovalently and with very little specificity to proteins. • Brilliant Blue G-250, also known as Coomassie Blue G. Brilliant Blue G-250 binds proteins nonspecifically through a large number of ionic and Van der Waals interactions. • The sizes of proteins in an extract can be calculated by comparing their migration to a set of standard proteins run on the same gel.
- 10. Thank you