Electrophoresis new1
•Download as PPTX, PDF•
1 like•492 views
This document discusses various electrophoretic techniques used to separate biomolecules like proteins and DNA. It describes the principles and procedures of agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), discontinuous PAGE, isoelectric focusing, and two-dimensional electrophoresis. These techniques separate molecules based on properties like their electrophoretic mobility, molecular weight, and isoelectric point within different types of gels and buffers under an applied electric field. The document also outlines solubilizers, detection methods, and applications of these electrophoretic procedures.
Report
Share
Report
Share
Recommended
Sds page gel electrophoresis
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.
Identification of proteins by 2D gel
Protein identification by 2D gels uses a combination of isoelectric focusing (IEF) and SDS-PAGE. In IEF, proteins are separated based on their isoelectric point using an acrylamide gel containing an ampholyte pH gradient established by an electric current. Next, SDS-PAGE further separates the proteins by size. Finally, the proteins are visualized after staining, allowing identification by their position on the 2D gel based on isoelectric point and molecular weight.
PAGE
PAGE (polyacrylamide gel electrophoresis) separates proteins by size through electrophoresis in a polyacrylamide gel. Proteins migrate through the gel at different rates depending on their size and charge, allowing separation. The polyacrylamide gel consists of a cross-linked polymer matrix formed from acrylamide monomers. There are two main types of gels - stacking gels and separating gels. Various techniques exist including native PAGE, SDS-PAGE, isoelectric focusing PAGE, and two-dimensional gel electrophoresis. SDS-PAGE and isoelectric focusing PAGE provide particularly high resolution separations.
SDS-PAGE Electrophoresis
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.
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.
2 d gel electrophoresis
Separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right-angle to one another.
Sodium Dodecyl Poly Acryl AMide Gel electrophoresis (SDS-PAGE)
Sodium Dodecyl Poly Acryl AMide Gel electrophoresis (SDS-PAGE)Marudhar Kesari Jain College for Women Vaniyambadi - 635 751, Tamil Nadu, INDIA.
This document describes the principle and process of SDS PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis), a technique used to separate proteins by size. SDS PAGE involves denaturing proteins with SDS detergent, running them through a polyacrylamide gel, and applying an electric current to separate the proteins by size. The document outlines the key steps, including sample preparation, gel preparation, running the gel, staining to visualize protein bands, and using protein standards to determine molecular weights of unknown proteins.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.
Recommended
Sds page gel electrophoresis
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.
Identification of proteins by 2D gel
Protein identification by 2D gels uses a combination of isoelectric focusing (IEF) and SDS-PAGE. In IEF, proteins are separated based on their isoelectric point using an acrylamide gel containing an ampholyte pH gradient established by an electric current. Next, SDS-PAGE further separates the proteins by size. Finally, the proteins are visualized after staining, allowing identification by their position on the 2D gel based on isoelectric point and molecular weight.
PAGE
PAGE (polyacrylamide gel electrophoresis) separates proteins by size through electrophoresis in a polyacrylamide gel. Proteins migrate through the gel at different rates depending on their size and charge, allowing separation. The polyacrylamide gel consists of a cross-linked polymer matrix formed from acrylamide monomers. There are two main types of gels - stacking gels and separating gels. Various techniques exist including native PAGE, SDS-PAGE, isoelectric focusing PAGE, and two-dimensional gel electrophoresis. SDS-PAGE and isoelectric focusing PAGE provide particularly high resolution separations.
SDS-PAGE Electrophoresis
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.
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.
2 d gel electrophoresis
Separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right-angle to one another.
Sodium Dodecyl Poly Acryl AMide Gel electrophoresis (SDS-PAGE)
Sodium Dodecyl Poly Acryl AMide Gel electrophoresis (SDS-PAGE)Marudhar Kesari Jain College for Women Vaniyambadi - 635 751, Tamil Nadu, INDIA.
This document describes the principle and process of SDS PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis), a technique used to separate proteins by size. SDS PAGE involves denaturing proteins with SDS detergent, running them through a polyacrylamide gel, and applying an electric current to separate the proteins by size. The document outlines the key steps, including sample preparation, gel preparation, running the gel, staining to visualize protein bands, and using protein standards to determine molecular weights of unknown proteins.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.
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.
SDS and 2D page
SDS-PAGE is a common method to separate proteins based on their molecular mass. The proteins are first denatured by SDS, which coats each protein with a uniform negative charge. This causes the proteins to migrate towards the anode during electrophoresis, with smaller proteins moving faster through the discontinuous gel system. Two-dimensional gel electrophoresis combines isoelectric focusing, which separates proteins based on isoelectric point, with SDS-PAGE to allow better separation of proteins with similar masses.
Polyacrylamide gel electrophoresis (PAGE)
Polyacrylamide gel electrophoresis (PAGE) allows researchers to separate proteins by size. Proteins are first denatured using heat and detergents to impart a uniform charge. They are then loaded into a discontinuous polyacrylamide gel system, where they are stacked and separated. The stacking gel prepares the proteins into a narrow band before entering the resolving gel, which separates the proteins based on size differences through a process of electrophoresis using buffer systems. After separation, proteins can be visualized on the gel by staining. PAGE is a powerful technique that provides high resolution to analyze protein samples.
Sds page electrophoresis
SDS-PAGE is a technique used to separate proteins .
The concept of stacking and resolving gel in this SDS-PAGE are briefly describe in very interesting and easy way to understand the role of stacking and resolving gel.
Two dimensional gel electrophoresis
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.
Electro phoresis
This document provides an overview of electrophoresis. It discusses how electrophoresis works, separating macromolecules like proteins and nucleic acids based on size and charge. Two main types of polyacrylamide gel electrophoresis are described: native PAGE and SDS PAGE. Native PAGE separates native proteins based on their charge, while SDS PAGE denatures proteins so they separate based solely on size. The document explains the principles and applications of electrophoresis techniques.
Running of SDS-PAGE Practical
Sepration of proteins on the basis of molecular weight.
SDS is used as denaturing agent along with Polyacrylamide gel.
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.
Sds page
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.
Two dimensional gel electrophoresis (2-DE)
Two-dimensional gel electrophoresis (2-DE) is considered a powerful tool for proteomics work. 2-DE separates proteins depending on two differ steps: the first one is called isoelectric focusing (IEF) which separates proteins according to isoelectric points (pI); the second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates proteins based on the molecular weights.
Our website: www.creative-proteomics.com
Koushik page electrophoresis
Electrophoresis is an analytical technique used to separate and characterize proteins, nucleic acids, and other particles based on their size and charge. It works by applying an electric field to move these molecules through a gel such as agarose or polyacrylamide. Different gel types and concentrations allow separation of molecules by size, including SDS-PAGE which uses SDS to denature proteins and separate them by mass. Electrophoresis has applications in molecular biology, medicine, quality control, and forensics.
2d sds page
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.
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.
ISOELECTRIC FOCUSING PPT - SLIDE SHARE
This document provides an overview of isoelectric focusing (IEF). IEF separates proteins in a gel according to their isoelectric point (pI), which is the pH at which a protein has no net charge. During IEF, proteins migrate through an immobilized pH gradient generated by ampholytes until they reach the pH that matches their pI and cease moving. IEF provides high resolution separation and is useful for research applications such as taxonomy, cytology, and immunology.
Sanjay
Gel electrophoresis is a technique used to separate biomolecules like DNA, RNA, or proteins based on their size and charge. It works by applying an electric current to a gel matrix, causing the charged molecules to migrate at different rates depending on their size and charge. There are two main types of gels used - agarose gels separate larger macromolecules, while polyacrylamide gels are used to separate smaller molecules like proteins due to their smaller pore size. The process involves casting the gel, loading samples and a molecular weight standard, running the gel at a constant voltage, and staining to visualize the separated biomolecules.
Trouble Shooting of SDS PAGE Analysis
This document provides information on troubleshooting SDS-PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis). It discusses common issues like sample preparation, gel casting, running conditions, and staining. Factors that can affect resolution include instrument assembly, gel preparation, sample nature/preparation, and run conditions. Specific problems covered include streaking, diffusion, bubbles, uneven fields, cracking, detachment, and faint bands. Solutions provided for resolving these issues.
Proteomics course 2
2D gel electrophoresis separates proteins based on their isoelectric point (pI) in the first dimension and molecular weight in the second dimension. Amino acids can exist as zwitterions and act as both acids and bases, donating or accepting protons. The isoelectric point is calculated using the Henderson-Hasselbalch equation and titration curves, taking into account the pKa values of amino acid side chains. Detection methods for proteins on 2D gels include staining with dyes or radioactive labeling.
Isoelectric focussing
Isoelectric focusing is a technique that separates proteins and other amphoteric molecules based on their isoelectric point, which is the pH at which they have no net charge. During isoelectric focusing, proteins are placed in an immobilized pH gradient formed by carrier ampholytes and an electric field is applied, causing the proteins to migrate through the pH gradient until they reach the pH that matches their isoelectric point and stop migrating. It is useful for analyzing protein heterogeneity and separating isoforms that have small charge differences but similar molecular weights.
Poly-acrylamide Gel Electrophoresis
This document discusses polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It defines gels and describes different types including organogels, xerogels, and hydrogels. It then focuses on polyacrylamide gels, explaining how they are made from acrylamide and bis-acrylamide monomers and can be used for electrophoresis. The document outlines the SDS-PAGE procedure, including how SDS treats proteins uniformly, and its applications in proteomics and ichthyotaxonomy.
SDS-PAGE
SDS-PAGE is a technique used to separate proteins by size. Proteins are treated with SDS to impart a uniform charge, then loaded into wells in a polyacrylamide gel. An electric current causes the proteins to migrate through the stacking gel and separating gel at rates inversely proportional to their log molecular weight. The separated proteins are then stained with Coomassie Brilliant Blue dye to visualize bands and determine their molecular weights by comparing to marker proteins.
Protein purification techniques
Protein purification techniquesDepartment of Biotechnology, Kamaraj college of engineering and technology
Dr.S.Karthikumar
Asst. Prof., Dept. of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India
skarthikumar@gmail.comGEL ELECTROPHORESIS
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.
More Related Content
What's hot
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.
SDS and 2D page
SDS-PAGE is a common method to separate proteins based on their molecular mass. The proteins are first denatured by SDS, which coats each protein with a uniform negative charge. This causes the proteins to migrate towards the anode during electrophoresis, with smaller proteins moving faster through the discontinuous gel system. Two-dimensional gel electrophoresis combines isoelectric focusing, which separates proteins based on isoelectric point, with SDS-PAGE to allow better separation of proteins with similar masses.
Polyacrylamide gel electrophoresis (PAGE)
Polyacrylamide gel electrophoresis (PAGE) allows researchers to separate proteins by size. Proteins are first denatured using heat and detergents to impart a uniform charge. They are then loaded into a discontinuous polyacrylamide gel system, where they are stacked and separated. The stacking gel prepares the proteins into a narrow band before entering the resolving gel, which separates the proteins based on size differences through a process of electrophoresis using buffer systems. After separation, proteins can be visualized on the gel by staining. PAGE is a powerful technique that provides high resolution to analyze protein samples.
Sds page electrophoresis
SDS-PAGE is a technique used to separate proteins .
The concept of stacking and resolving gel in this SDS-PAGE are briefly describe in very interesting and easy way to understand the role of stacking and resolving gel.
Two dimensional gel electrophoresis
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.
Electro phoresis
This document provides an overview of electrophoresis. It discusses how electrophoresis works, separating macromolecules like proteins and nucleic acids based on size and charge. Two main types of polyacrylamide gel electrophoresis are described: native PAGE and SDS PAGE. Native PAGE separates native proteins based on their charge, while SDS PAGE denatures proteins so they separate based solely on size. The document explains the principles and applications of electrophoresis techniques.
Running of SDS-PAGE Practical
Sepration of proteins on the basis of molecular weight.
SDS is used as denaturing agent along with Polyacrylamide gel.
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.
Sds page
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.
Two dimensional gel electrophoresis (2-DE)
Two-dimensional gel electrophoresis (2-DE) is considered a powerful tool for proteomics work. 2-DE separates proteins depending on two differ steps: the first one is called isoelectric focusing (IEF) which separates proteins according to isoelectric points (pI); the second step is SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which separates proteins based on the molecular weights.
Our website: www.creative-proteomics.com
Koushik page electrophoresis
Electrophoresis is an analytical technique used to separate and characterize proteins, nucleic acids, and other particles based on their size and charge. It works by applying an electric field to move these molecules through a gel such as agarose or polyacrylamide. Different gel types and concentrations allow separation of molecules by size, including SDS-PAGE which uses SDS to denature proteins and separate them by mass. Electrophoresis has applications in molecular biology, medicine, quality control, and forensics.
2d sds page
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.
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.
ISOELECTRIC FOCUSING PPT - SLIDE SHARE
This document provides an overview of isoelectric focusing (IEF). IEF separates proteins in a gel according to their isoelectric point (pI), which is the pH at which a protein has no net charge. During IEF, proteins migrate through an immobilized pH gradient generated by ampholytes until they reach the pH that matches their pI and cease moving. IEF provides high resolution separation and is useful for research applications such as taxonomy, cytology, and immunology.
Sanjay
Gel electrophoresis is a technique used to separate biomolecules like DNA, RNA, or proteins based on their size and charge. It works by applying an electric current to a gel matrix, causing the charged molecules to migrate at different rates depending on their size and charge. There are two main types of gels used - agarose gels separate larger macromolecules, while polyacrylamide gels are used to separate smaller molecules like proteins due to their smaller pore size. The process involves casting the gel, loading samples and a molecular weight standard, running the gel at a constant voltage, and staining to visualize the separated biomolecules.
Trouble Shooting of SDS PAGE Analysis
This document provides information on troubleshooting SDS-PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis). It discusses common issues like sample preparation, gel casting, running conditions, and staining. Factors that can affect resolution include instrument assembly, gel preparation, sample nature/preparation, and run conditions. Specific problems covered include streaking, diffusion, bubbles, uneven fields, cracking, detachment, and faint bands. Solutions provided for resolving these issues.
Proteomics course 2
2D gel electrophoresis separates proteins based on their isoelectric point (pI) in the first dimension and molecular weight in the second dimension. Amino acids can exist as zwitterions and act as both acids and bases, donating or accepting protons. The isoelectric point is calculated using the Henderson-Hasselbalch equation and titration curves, taking into account the pKa values of amino acid side chains. Detection methods for proteins on 2D gels include staining with dyes or radioactive labeling.
Isoelectric focussing
Isoelectric focusing is a technique that separates proteins and other amphoteric molecules based on their isoelectric point, which is the pH at which they have no net charge. During isoelectric focusing, proteins are placed in an immobilized pH gradient formed by carrier ampholytes and an electric field is applied, causing the proteins to migrate through the pH gradient until they reach the pH that matches their isoelectric point and stop migrating. It is useful for analyzing protein heterogeneity and separating isoforms that have small charge differences but similar molecular weights.
Poly-acrylamide Gel Electrophoresis
This document discusses polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). It defines gels and describes different types including organogels, xerogels, and hydrogels. It then focuses on polyacrylamide gels, explaining how they are made from acrylamide and bis-acrylamide monomers and can be used for electrophoresis. The document outlines the SDS-PAGE procedure, including how SDS treats proteins uniformly, and its applications in proteomics and ichthyotaxonomy.
SDS-PAGE
SDS-PAGE is a technique used to separate proteins by size. Proteins are treated with SDS to impart a uniform charge, then loaded into wells in a polyacrylamide gel. An electric current causes the proteins to migrate through the stacking gel and separating gel at rates inversely proportional to their log molecular weight. The separated proteins are then stained with Coomassie Brilliant Blue dye to visualize bands and determine their molecular weights by comparing to marker proteins.
What's hot (20)
Similar to Electrophoresis new1
Protein purification techniques
Protein purification techniquesDepartment of Biotechnology, Kamaraj college of engineering and technology
Dr.S.Karthikumar
Asst. Prof., Dept. of Biotechnology
Kamaraj College of Engineering and Technology
S.P.G.C.Nagar, Virudhunagar, Tamilnadu, India
skarthikumar@gmail.comGEL ELECTROPHORESIS
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.
gel electrophoresis
Gel electrophoresis is a technique used to separate biomolecules like proteins and nucleic acids based on their charge and size. It works by applying an electric field to move charged molecules through a gel towards the electrode of opposite charge. There are different types of gels used like agarose, polyacrylamide and starch depending on the size of molecules to be separated. Samples are loaded into wells in the gel and the electric current causes smaller molecules to migrate farther. After separation, the molecules can be visualized using dyes and the technique has various applications in fields like forensics, molecular biology and biochemistry.
Zone electrophoresis and its types
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.
Gel electrophoresis
Gel electrophoresis is a technique used to separate biomolecules like proteins, nucleic acids, and other charged molecules. It works by applying an electric field to move these charged molecules through a gel based on their size and charge. There are different types of gels used like agarose, polyacrylamide, and starch depending on the size of molecules to be separated. Samples are loaded into wells in the gel and an electric current is applied causing molecules to migrate through the gel at different rates. The separated biomolecules can then be visualized based on properties like fluorescence or staining to analyze differences in molecular weights and sizes. Gel electrophoresis has many applications in fields like forensics, molecular biology, genetics, and biochemistry
Electrophoresis
Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules based on their size and electrical charge. An electric current is used to move molecules to be separated through a gel. Pores in the gel work like a sieve, allowing smaller molecules to move faster than larger molecules.
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.
Isoelectric Focusing.pptx
Macro molecule separation technique in biochemistry. PAGE; Isoelectric Focusing. DNA AND RNA separation techniques. nucleotide separation techniques.
Essentials of Gel Electrophoresis
This lecture is about Gel Electrophoresis and a little brief about it, which is presented by Tuba Nafees she is MSc graduate in Biotechnology from University of Karachi, Sindh Pakistan.
For youtube :https://www.youtube.com/watch?v=G4dwvDkxKN4
Electrophoresis
Electrophoresis is an electrokinetic process which separates charged particles in a fluid using a field of electrical charge. It is most often used in life sciences to separate protein molecules or DNA and can be achieved through several different procedures depending on the type and size of the molecules. The procedures differ in some ways but all need a source for the electrical charge, a support medium and a buffer solution. Electrophoresis is used in laboratories for the separation of molecules based on size, density and purity.An electric field is applied to molecules and as they are electrically charged themselves it results in a force acting upon them. The greater the charge of the molecule the greater the force applied by the electrical field and therefore the further through the support medium the molecule will move relative to its mass.
Some example applications of electrophoresis include DNA and RNA analysis as well as protein electrophoresis which is a medical procedure used to analyse and separate the molecules found in a fluid sample (most commonly blood and urine samples).Different types of gels are usually used as the support medium for electrophoresis and this may be in slab or tube form depending on which is more beneficial. Gel slabs enable many samples to be run simultaneously and so are frequently used in laboratories. However, tube gels give a better resolution of the results so are often chosen for protein electrophoresis.
Agarose gel is commonly used for electrophoresis of DNA. It has a large pore structure allowing larger molecules to move easily but it is not suitable for sequencing smaller molecules.
Polyacrylamide gel electrophoresis (PAGE) has a clearer resolution than agarose gel making it more suitable for quantitative analysis. This makes it possible to identify how proteins bind to DNA. It can also be used to develop an understanding of how bacteria is becoming resistant to antibiotics through plasmid analysis.
Vertical Gel Electrophoresis (SDS-PAGE)
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.
Gel electrophoresis
The document provides information on gel electrophoresis techniques. It discusses different types of gels like agarose, polyacrylamide, and starch that can be used for electrophoresis. Agarose gel is commonly used to separate macromolecules like nucleic acids and proteins due to its large pore size. Polyacrylamide gel is used to separate smaller molecules and provides better resolution than agarose. SDS-PAGE allows separation based on molecular weight by making all proteins negatively charged. The document outlines the procedures for agarose and polyacrylamide gel electrophoresis and their applications in analyzing proteins and nucleic acids.
Nanofiltration&electrophoresis
Nanfiltration is a membrane filtration process that rejects molecules around 1 nanometer in size. It can separate inorganic and organic substances from liquid. Nanfiltration uses semi-permeable membranes under pressure to filter water. Electrophoresis is a technique that separates ions based on their migration in an electric field. Positively charged ions migrate toward the negative electrode and negatively charged ions migrate toward the positive electrode. Common electrophoresis methods include slab gel electrophoresis and capillary electrophoresis.
ELECTROPHORESIS kaif.pptx
electrophoresis: movement of charge particles in a gel under the influence of an electric field, principle, factors, apparatus, types , application, advantage and disadvantage.
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.
Polyacrylamide gel electrophoresis
i am HAFIZ M WASEEM from mailsi vehari
BSc in science college Multan Pakistan
MSC university of education Lahore Pakistan
I love Pakistan and my teachers
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
Electrophoresis
Electrophoresis is a method used to separate charged particles such as proteins and nucleic acids based on differences in their migration speed in an electric field. There are several types of electrophoresis including agarose gel electrophoresis, SDS-PAGE, isoelectric focusing, capillary electrophoresis, and two-dimensional electrophoresis. During electrophoresis, charged particles migrate through a gel or capillary towards an electrode with the rate of migration depending on factors like the particle's charge, size, and shape.
Polyacrylamidegelelectrophoresis PAGE
Polyacrylamide gel electrophoresis (PAGE) is a technique used to separate proteins based on their size and charge. PAGE uses a polyacrylamide gel with a tight matrix and small pore sizes, allowing for the separation of smaller proteins. Sodium dodecyl sulfate-PAGE (SDS-PAGE) is a common type that uses the detergent SDS to denature proteins and impart a uniform negative charge, allowing separation based solely on molecular weight. The gel consists of a stacking gel that concentrates proteins, and a resolving gel where separation occurs. Proteins are visualized after electrophoresis by staining.
96481966-Electrophoresis.ppt
This document provides an overview of electrophoresis techniques. It defines electrophoresis as the migration of charged solutes in a liquid medium under an electrical field. It then discusses various electrophoresis techniques including zone electrophoresis, isotachophoresis, agarose gel electrophoresis, cellulose acetate electrophoresis, polyacrylamide gel electrophoresis, isoelectric focusing, and two-dimensional electrophoresis. It provides details on the principles, methods, applications and considerations for each technique.
Similar to Electrophoresis new1 (20)
Recently uploaded
Candidate young stellar objects in the S-cluster: Kinematic analysis of a sub...
Context. The observation of several L-band emission sources in the S cluster has led to a rich discussion of their nature. However, a definitive answer to the classification of the dusty objects requires an explanation for the detection of compact Doppler-shifted Brγ emission. The ionized hydrogen in combination with the observation of mid-infrared L-band continuum emission suggests that most of these sources are embedded in a dusty envelope. These embedded sources are part of the S-cluster, and their relationship to the S-stars is still under debate. To date, the question of the origin of these two populations has been vague, although all explanations favor migration processes for the individual cluster members. Aims. This work revisits the S-cluster and its dusty members orbiting the supermassive black hole SgrA* on bound Keplerian orbits from a kinematic perspective. The aim is to explore the Keplerian parameters for patterns that might imply a nonrandom distribution of the sample. Additionally, various analytical aspects are considered to address the nature of the dusty sources. Methods. Based on the photometric analysis, we estimated the individual H−K and K−L colors for the source sample and compared the results to known cluster members. The classification revealed a noticeable contrast between the S-stars and the dusty sources. To fit the flux-density distribution, we utilized the radiative transfer code HYPERION and implemented a young stellar object Class I model. We obtained the position angle from the Keplerian fit results; additionally, we analyzed the distribution of the inclinations and the longitudes of the ascending node. Results. The colors of the dusty sources suggest a stellar nature consistent with the spectral energy distribution in the near and midinfrared domains. Furthermore, the evaporation timescales of dusty and gaseous clumps in the vicinity of SgrA* are much shorter ( 2yr) than the epochs covered by the observations (≈15yr). In addition to the strong evidence for the stellar classification of the D-sources, we also find a clear disk-like pattern following the arrangements of S-stars proposed in the literature. Furthermore, we find a global intrinsic inclination for all dusty sources of 60 ± 20◦, implying a common formation process. Conclusions. The pattern of the dusty sources manifested in the distribution of the position angles, inclinations, and longitudes of the ascending node strongly suggests two different scenarios: the main-sequence stars and the dusty stellar S-cluster sources share a common formation history or migrated with a similar formation channel in the vicinity of SgrA*. Alternatively, the gravitational influence of SgrA* in combination with a massive perturber, such as a putative intermediate mass black hole in the IRS 13 cluster, forces the dusty objects and S-stars to follow a particular orbital arrangement. Key words. stars: black holes– stars: formation– Galaxy: center– galaxies: star formation
Gadgets for management of stored product pests_Dr.UPR.pdf
Insectsplayamajorroleinthedeteriorationoffoodgrainscausingbothquantitativeandqualitativelosses
Wellprovedthatnogranariescanbefilledwithgrainswithoutinsectsastheharvestedproducecontainegg(or)larvae(or)pupae(or)adultinsectinthembecauseoffieldcarryoverinfestationwhichcannotbeavoidedindevelopingcountrieslikeIndia
Simpletechnologiesfortimelydetectionofinsectsinthestoredproduceandtherebyplantimelycontrolmeasures
Mending Clothing to Support Sustainable Fashion_CIMaR 2024.pdf
Ozturkcan, S., Berndt, A., & Angelakis, A. (2024). Mending clothing to support sustainable fashion. Presented at the 31st Annual Conference by the Consortium for International Marketing Research (CIMaR), 10-13 Jun 2024, University of Gävle, Sweden.
Injection: Risks and challenges - Injection of CO2 into geological rock forma...
Presentation by Dr. Florian Krob, Workshop "Risks of CO2 storage - How can they be addressed and reduced?", 22.4.2024
Holsinger, Bruce W. - Music, body and desire in medieval culture [2001].pdf
Music and Medieval History
TOPIC OF DISCUSSION: CENTRIFUGATION SLIDESHARE.pptx
Centrifugation is a powerful technique used in laboratories to separate components of a heterogeneous mixture based on their density. This process utilizes centrifugal force to rapidly spin samples, causing denser particles to migrate outward more quickly than lighter ones. As a result, distinct layers form within the sample tube, allowing for easy isolation and purification of target substances.
Anti-Universe And Emergent Gravity and the Dark Universe
Recent theoretical progress indicates that spacetime and gravity emerge together from the entanglement structure of an underlying microscopic theory. These ideas are best understood in Anti-de Sitter space, where they rely on the area law for entanglement entropy. The extension to de Sitter space requires taking into account the entropy and temperature associated with the cosmological horizon. Using insights from string theory, black hole physics and quantum information theory we argue that the positive dark energy leads to a thermal volume law contribution to the entropy that overtakes the area law precisely at the cosmological horizon. Due to the competition between area and volume law entanglement the microscopic de Sitter states do not thermalise at sub-Hubble scales: they exhibit memory effects in the form of an entropy displacement caused by matter. The emergent laws of gravity contain an additional ‘dark’ gravitational force describing the ‘elastic’ response due to the entropy displacement. We derive an estimate of the strength of this extra force in terms of the baryonic mass, Newton’s constant and the Hubble acceleration scale a0 = cH0, and provide evidence for the fact that this additional ‘dark gravity force’ explains the observed phenomena in galaxies and clusters currently attributed to dark matter.
Embracing Deep Variability For Reproducibility and Replicability
Embracing Deep Variability For Reproducibility and ReplicabilityUniversity of Rennes, INSA Rennes, Inria/IRISA, CNRS
Embracing Deep Variability For Reproducibility and Replicability
Abstract: Reproducibility (aka determinism in some cases) constitutes a fundamental aspect in various fields of computer science, such as floating-point computations in numerical analysis and simulation, concurrency models in parallelism, reproducible builds for third parties integration and packaging, and containerization for execution environments. These concepts, while pervasive across diverse concerns, often exhibit intricate inter-dependencies, making it challenging to achieve a comprehensive understanding. In this short and vision paper we delve into the application of software engineering techniques, specifically variability management, to systematically identify and explicit points of variability that may give rise to reproducibility issues (eg language, libraries, compiler, virtual machine, OS, environment variables, etc). The primary objectives are: i) gaining insights into the variability layers and their possible interactions, ii) capturing and documenting configurations for the sake of reproducibility, and iii) exploring diverse configurations to replicate, and hence validate and ensure the robustness of results. By adopting these methodologies, we aim to address the complexities associated with reproducibility and replicability in modern software systems and environments, facilitating a more comprehensive and nuanced perspective on these critical aspects.
https://hal.science/hal-04582287
Post translation modification by Suyash Garg
overview of PTM helps to the students who wants to clear their basics about it.
Male reproduction physiology by Suyash Garg .pptx
Physiology of Male reproduction.
Video mentioned at page no. 23 as summary for better understanding
JAMES WEBB STUDY THE MASSIVE BLACK HOLE SEEDS
The pathway(s) to seeding the massive black holes (MBHs) that exist at the heart of galaxies in the present and distant Universe remains an unsolved problem. Here we categorise, describe and quantitatively discuss the formation pathways of both light and heavy seeds. We emphasise that the most recent computational models suggest that rather than a bimodal-like mass spectrum between light and heavy seeds with light at one end and heavy at the other that instead a continuum exists. Light seeds being more ubiquitous and the heavier seeds becoming less and less abundant due the rarer environmental conditions required for their formation. We therefore examine the different mechanisms that give rise to different seed mass spectrums. We show how and why the mechanisms that produce the heaviest seeds are also among the rarest events in the Universe and are hence extremely unlikely to be the seeds for the vast majority of the MBH population. We quantify, within the limits of the current large uncertainties in the seeding processes, the expected number densities of the seed mass spectrum. We argue that light seeds must be at least 103 to 105 times more numerous than heavy seeds to explain the MBH population as a whole. Based on our current understanding of the seed population this makes heavy seeds (Mseed > 103 M⊙) a significantly more likely pathway given that heavy seeds have an abundance pattern than is close to and likely in excess of 10−4 compared to light seeds. Finally, we examine the current state-of-the-art in numerical calculations and recent observations and plot a path forward for near-future advances in both domains.
Methods of grain storage Structures in India.pdf
•Post-harvestlossesaccountforabout10%oftotalfoodgrainsduetounscientificstorage,insects,rodents,micro-organismsetc.,
•Totalfoodgrainproductioninindiais311milliontonnesandstorageis145mt.InIndia,annualstoragelosseshavebeenestimated14mtworthofRs.7,000croreinwhichinsectsaloneaccountfornearlyRs.1,300crores.
•InIndiaoutofthetotalproduction,about30%ismarketablesurplus
•Remaining70%isretainedandstoredbyfarmersforconsumption,seed,feed.Hence,growerneedstoragefacilitytoholdaportionofproducetosellwhenthemarketingpriceisfavourable
•TradersandCo-operativesatmarketcentresneedstoragestructurestoholdgrainswhenthetransportfacilityisinadequate
Farming systems analysis: what have we learnt?.pptx
Presentation given at the official farewell of Prof Ken Gillet at Wageningen on 13 June 2024
Pests of Storage_Identification_Dr.UPR.pdf
InIndia-post-harvestlosses-unscientificstorage,insects,rodents,micro-organismsetc.,accountforabout10percentoftotalfoodgrains
Graininfestation
Directdamage
Indirectly
•theexuviae,skin,deadinsects
•theirexcretawhichmakefoodunfitforhumanconsumption
About600speciesofinsectshavebeenassociatedwithstoredgrainproducts
100speciesofinsectpestsofstoredproductscauseeconomiclosses
Recently uploaded (20)
Candidate young stellar objects in the S-cluster: Kinematic analysis of a sub...
Candidate young stellar objects in the S-cluster: Kinematic analysis of a sub...
Gadgets for management of stored product pests_Dr.UPR.pdf
Gadgets for management of stored product pests_Dr.UPR.pdf
Mending Clothing to Support Sustainable Fashion_CIMaR 2024.pdf
Mending Clothing to Support Sustainable Fashion_CIMaR 2024.pdf
Immunotherapy presentation from clinical immunology
Immunotherapy presentation from clinical immunology
Injection: Risks and challenges - Injection of CO2 into geological rock forma...
Injection: Risks and challenges - Injection of CO2 into geological rock forma...
Holsinger, Bruce W. - Music, body and desire in medieval culture [2001].pdf
Holsinger, Bruce W. - Music, body and desire in medieval culture [2001].pdf
TOPIC OF DISCUSSION: CENTRIFUGATION SLIDESHARE.pptx
TOPIC OF DISCUSSION: CENTRIFUGATION SLIDESHARE.pptx
Anti-Universe And Emergent Gravity and the Dark Universe
Anti-Universe And Emergent Gravity and the Dark Universe
Embracing Deep Variability For Reproducibility and Replicability
Embracing Deep Variability For Reproducibility and Replicability
Microbiology of Central Nervous System INFECTIONS.pdf
Microbiology of Central Nervous System INFECTIONS.pdf
Farming systems analysis: what have we learnt?.pptx
Farming systems analysis: what have we learnt?.pptx
Electrophoresis new1
- 4. Electrophoretic mobility in the gel logE = logE’- KrG •E- electrophoretic mobility •E’- mobility in sucrose solution •Kr- retardation coefficient •G- Gel concentration
- 5. Solubilizers • Urea- conc. 3-12M, Disrupt Hydrogen bonds • SDS- Anionic detergent, imparts negative charge, Disturbs hydrophobic interactions. • CTAB- Cationic detergent. • B- mercaptoethanol- Distrubs disulphide linkage
- 11. Agarose gel electrophoresis • Structure of Agarose gel • Preparation of gel • Submerged gel electrophoresis • Detection • Fluorescent method for NA. • Staining for proteins
- 12. Structure of Agarose Gel
- 14. PAGE • Structure of gel • Components of gel • Electrophoretic run • Detection
- 22. Applications • Separation of proteins • DNA Sequencing • Western Blotting • Determination of molecular weight of proteins
- 24. Isoelectric Focussing • Introduction • Principle • Establishing the pH gradient- carrier ampholytes • Stabilization against Convection • Procedure • Separation of protein from carrier ampholytes • Applications
- 27. Determination of isoelectric Point of a protein • Measure the mobility of the protein at several pH values. • Plot mobility values against the pH values. • Plot intercept at zero mobility.
- 28. Establishing the pH gradient- carrier ampholytes • Isomers and homologs of aliphatic polyamino polycarboxylic acids. • R- N-(CH2)n- N - (CH2)n- COOH Where R- (CH2)n- COOH, H • n- less than 5 • Ex.- ampholine, Pharmalyte, Bio-lyte. • Used in 1% concentration
- 29. Properties • Carrier ampholytes must dictate the pH course, should have a certain buffering capacity at their isoelectric point. • Should have a conductance at their isoelectric point. • Low molecular weight. • Should be soluble in water. • Should have law absorption at 280nm.
- 30. Stabilization against convection 1)Density Gradient- • Uncharged solutes dissolvable in water • Should not react with proteins, low metal content, high purity • Ex.- sucrose- 50% , up to pH 10.protective action on protein. • Glycerol, ficoll, sorbitol, ethylene glycol, dextran 2)Gel- as anticonvectant, 7.5% acrylamide • High molecular weight protein- .5%
- 32. Procedure- Column- density gradient Plate- polyacrylamide gel
- 33. Separation of protein from carrier ampholytes • Avg. mol.wt of ampholyte- 800D, • Avg. mol.wt of protein- 10000D • Method of separation – 1) Dialysis – 99% efficient, Slow process 2) Gel filtration – Sephadex G-50 3) Ammonium sulphate precipitation 4)Ion exchange chromatography 5) Partition chromatography
- 34. Applications • Separation and identification of serum proteins. • Used in food and agricultural industries. • Forensic & human genetics labs • Research in enzymology, immunology & membrane biochemistry
- 35. Two dimensional electrophoresis • Combination of isoelectric focussing & SDS-PAGE. • Can resolve 5000 proteins in individual bands. • Uses isoelectric pH and molecular weight combination