Western blotting WB (immunoblotting) is a widely practiced analytical technique to detect target proteins within samples using antigen-specific antibodies.
This document provides an overview of the principles and procedures of western blotting. It discusses:
1) Protein extraction and quantitation from cell and tissue samples.
2) Separation of proteins by electrophoresis using SDS-PAGE gels to separate based on molecular weight.
3) Transfer of separated proteins from the gel onto a membrane using wet or semi-dry transfer methods.
4) Blocking of the membrane to reduce nonspecific binding before primary antibody incubation.
5) Detection of target proteins using enzyme-conjugated secondary antibodies and chromogenic or chemiluminescent substrates.
Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. ... Zymography also refers to a collection of related, fermented products, considered as a body of work.
Serum protein electrophoresis & their clinical importanceDr.M.Prasad Naidu
This document discusses serum proteins and electrophoresis techniques used to analyze them. It provides details on the major serum proteins - albumin and globulins - and their functions. Electrophoresis separates proteins based on their charge and size. Several electrophoresis methods are described, including agarose gel, SDS-PAGE, and capillary electrophoresis. Factors influencing electrophoresis results and common stains used are also outlined. The document concludes with descriptions of normal and abnormal serum protein electrophoresis patterns and their clinical significance.
Protein Detection Methods and Applicationangelsalaman
The document discusses various protein analysis methods including gel electrophoresis, SDS-PAGE, and native gel electrophoresis. Gel electrophoresis separates proteins, DNA, and RNA using an electric current applied to a gel matrix. SDS-PAGE separates denatured proteins by size, coating them with negative charges. Native gel electrophoresis separates intact proteins by their intrinsic charge and hydrodynamic size, allowing analysis of conformation, aggregation, and binding events.
This document discusses methods for expressing and purifying recombinant proteins. It describes commonly used vectors for inserting genes of interest, such as plasmids and artificial chromosomes. The basic steps are outlined as amplifying the gene, inserting it into a cloning vector, subcloning into an expression vector, transforming the vector into a protein-expressing organism, and testing for protein identification. Methods for isolating and purifying the protein include cell disruption, centrifugation, concentration techniques, and differential centrifugation. Common purification methods described are based on separating proteins by charge, size, hydrophobicity, and specific binding sites using techniques like ion exchange chromatography, size exclusion chromatography, and affinity chromatography.
This document discusses advanced electrophoresis techniques used in genomics and proteomics. It describes how electrophoresis separates charged molecules like DNA, RNA, and proteins based on their size, shape, and charge. It then explains different supporting media like cellulose acetate, agarose, and polyacrylamide gels and how they separate molecules. The document also covers techniques like two-dimensional electrophoresis, pulsed field gel electrophoresis, isoelectric focusing, capillary electrophoresis, and methods to detect DNA polymorphisms and mutations.
The document summarizes strategies for protein purification. It discusses that protein purification separates and isolates proteins from complex mixtures using differences in their physical and chemical properties. It outlines various centrifugation and chromatography techniques used in protein purification, including differential centrifugation, gel filtration, ion exchange chromatography, and affinity chromatography. These techniques separate proteins based on properties like size, charge, and binding affinity. The document also notes that protein purification is now performed from research to industrial scales and that affinity tagging has revolutionized the field.
Electrophoresis principle and types by Dr. Anurag YadavDr Anurag Yadav
the general principle on how the electrophoresis performs.
the different types of electrophoresis and the mechanism of separation based on different character of the medium and type of electrophoresis.
This document provides an overview of the principles and procedures of western blotting. It discusses:
1) Protein extraction and quantitation from cell and tissue samples.
2) Separation of proteins by electrophoresis using SDS-PAGE gels to separate based on molecular weight.
3) Transfer of separated proteins from the gel onto a membrane using wet or semi-dry transfer methods.
4) Blocking of the membrane to reduce nonspecific binding before primary antibody incubation.
5) Detection of target proteins using enzyme-conjugated secondary antibodies and chromogenic or chemiluminescent substrates.
Zymography is an electrophoretic technique for the detection of hydrolytic enzymes, based on the substrate repertoire of the enzyme. ... Zymography also refers to a collection of related, fermented products, considered as a body of work.
Serum protein electrophoresis & their clinical importanceDr.M.Prasad Naidu
This document discusses serum proteins and electrophoresis techniques used to analyze them. It provides details on the major serum proteins - albumin and globulins - and their functions. Electrophoresis separates proteins based on their charge and size. Several electrophoresis methods are described, including agarose gel, SDS-PAGE, and capillary electrophoresis. Factors influencing electrophoresis results and common stains used are also outlined. The document concludes with descriptions of normal and abnormal serum protein electrophoresis patterns and their clinical significance.
Protein Detection Methods and Applicationangelsalaman
The document discusses various protein analysis methods including gel electrophoresis, SDS-PAGE, and native gel electrophoresis. Gel electrophoresis separates proteins, DNA, and RNA using an electric current applied to a gel matrix. SDS-PAGE separates denatured proteins by size, coating them with negative charges. Native gel electrophoresis separates intact proteins by their intrinsic charge and hydrodynamic size, allowing analysis of conformation, aggregation, and binding events.
This document discusses methods for expressing and purifying recombinant proteins. It describes commonly used vectors for inserting genes of interest, such as plasmids and artificial chromosomes. The basic steps are outlined as amplifying the gene, inserting it into a cloning vector, subcloning into an expression vector, transforming the vector into a protein-expressing organism, and testing for protein identification. Methods for isolating and purifying the protein include cell disruption, centrifugation, concentration techniques, and differential centrifugation. Common purification methods described are based on separating proteins by charge, size, hydrophobicity, and specific binding sites using techniques like ion exchange chromatography, size exclusion chromatography, and affinity chromatography.
This document discusses advanced electrophoresis techniques used in genomics and proteomics. It describes how electrophoresis separates charged molecules like DNA, RNA, and proteins based on their size, shape, and charge. It then explains different supporting media like cellulose acetate, agarose, and polyacrylamide gels and how they separate molecules. The document also covers techniques like two-dimensional electrophoresis, pulsed field gel electrophoresis, isoelectric focusing, capillary electrophoresis, and methods to detect DNA polymorphisms and mutations.
The document summarizes strategies for protein purification. It discusses that protein purification separates and isolates proteins from complex mixtures using differences in their physical and chemical properties. It outlines various centrifugation and chromatography techniques used in protein purification, including differential centrifugation, gel filtration, ion exchange chromatography, and affinity chromatography. These techniques separate proteins based on properties like size, charge, and binding affinity. The document also notes that protein purification is now performed from research to industrial scales and that affinity tagging has revolutionized the field.
Electrophoresis principle and types by Dr. Anurag YadavDr Anurag Yadav
the general principle on how the electrophoresis performs.
the different types of electrophoresis and the mechanism of separation based on different character of the medium and type of electrophoresis.
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.
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.
Gel Based Proteomics and Protein Sequences AnalysisGelica F
Two-dimensional gel electrophoresis (2DE) is the standard method for quantitative proteome analysis. It combines protein separation based on isoelectric focusing and molecular weight. In the first dimension, proteins are separated based on their isoelectric point using immobilized pH gradients. In the second dimension, proteins are separated by molecular weight using SDS-PAGE. The separated protein spots are then analyzed using mass spectrometry to identify individual proteins. 2DE provides high resolution and the ability to analyze thousands of proteins simultaneously, but it also has limitations including irreproducibility and inability to resolve all proteins.
Analytical techniques for separation or purification of proteinsrohini sane
A comprehensive presentation on Analytical techniques for separation or purification of proteins for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.
2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.
Electrophoresis is a technique used to separate charged biomolecules like proteins and nucleic acids. It works by applying an electric field to migrate these molecules through a buffer solution or gel based on their charge to mass ratio. The first major use of electrophoresis was in 1937. Key aspects include using a buffer to maintain pH and conductivity, as well as staining and destaining techniques to visualize separated biomolecules. Common types include gel electrophoresis using agarose or polyacrylamide gels as well as paper electrophoresis. Applications include clinical diagnosis and protein research.
Proteins – Basics you need to know for ProteomicsLionel Wolberger
The document provides an overview of key concepts in proteomics, including:
1) It discusses protein structure and function, the 20 common amino acids, and post-translational modifications that proteins undergo.
2) It introduces common techniques used in proteomics like chromatography, electrophoresis, mass spectrometry, and bioinformatics.
3) It summarizes protein analysis methods like gel electrophoresis, isoelectric focusing, and immunological assays used to detect and purify proteins of interest.
This document provides an overview of various protein purification techniques, including:
- Chromatography methods like affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography.
- Key steps in protein purification like cell lysis, centrifugation, assaying fractions for protein and activity, and monitoring purity with SDS-PAGE gels.
- Considerations for each chromatography method like matrix selection, binding capacities, and driving forces.
- Emerging techniques like capillary electrochromatography and multi-dimensional separations.
The document discusses proteomics, which is the study of the entire complement of proteins in a cell or organism. It defines key proteomics terms like proteome and describes techniques used in proteomics like protein separation, 2D gel electrophoresis, mass spectrometry, and protein digestion. The goals of proteomics include detecting and comparing protein expression profiles to understand biological processes and discover drug targets. Proteomics provides important insights not available through genomics alone.
Electrophoresis is a method used to separate macromolecules like DNA, RNA, and proteins based on their size and charge. There are several electrophoretic techniques including gel electrophoresis using agarose, polyacrylamide, or gradient gels, as well as capillary electrophoresis, isoelectric focusing, and microchip electrophoresis. Two-dimensional gel electrophoresis separates proteins in two steps based on isoelectric point and molecular weight to analyze complex protein mixtures.
This study used zymography to detect proteolytic enzymes in wheat leaves. One-dimensional (1D) zymography revealed 7 bands of proteolytic activity, which were assigned to proteinase families using specific inhibitors. Two-dimensional (2D) zymography provided additional separation of proteases according to isoelectric point, revealing multiple isoforms within families. Metalloproteinases were detected as a 150 kDa band inhibited by EDTA. Aspartic proteinases appeared as a 115-118 kDa double band inhibited by pepstatin. Serine and cysteine proteinases of various sizes were also observed. 2D zymography enabled visualization of isoforms within proteinase families.
The document discusses purification of recombinant proteins using affinity tags. It describes immobilized metal affinity chromatography (IMAC) as a widely used method to purify recombinant proteins fused to tags like histidine, GST or MBP. The document outlines the steps involved, including gene amplification, cloning, expression in bacteria or yeast, and purification. It focuses on using histidine tags and nickel-chelate affinity chromatography, noting the advantages of tags for simplifying purification and detection of recombinant proteins.
This document provides an overview of analytical proteomics and its applications. It defines the proteome and proteomics, and discusses the difference between protein biochemistry and proteomics. It describes the key steps in analytical proteomics: protein separation techniques like 1D/2D gel electrophoresis and isoelectric focusing; protein digestion using proteases like trypsin; and protein identification via mass spectrometry. Finally, it outlines some applications of proteomics like comparing proteomes under different conditions to study biological processes.
The document discusses aptamers, which are oligonucleotides or peptides that bind to targets with high affinity and specificity due to their unique three-dimensional structures. It describes how aptamers are produced through an in vitro selection process called SELEX and classified based on their structure and selection technique. The document also compares aptamers to antibodies and outlines several applications of aptamers in therapeutics, drug delivery, bioimaging, diagnostics, and more.
Scott Malcolm | Describe About Purpose and Creation Process of Gel Electropho...Scott Malcolm Dallas
Scott Malcolm is a great businessman, who lives in Dallas, Taxes. He explains here about biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length and to estimate the size of DNA and RNA fragments.
This document summarizes techniques for exploring and analyzing proteins, including concentrating purified proteins using lyophilization or ultrafiltration, separating proteins using electrophoresis or mass spectrometry, and identifying proteins using mass spectrometry. Electrophoresis techniques like SDS-PAGE and 2D gels separate proteins based on size and charge, allowing visualization and quantification of purified proteins. Mass spectrometry further identifies proteins by correlating detected ion masses with known protein standards. These techniques provide a quantitative evaluation of protein purification schemes.
Polyacrylamide gel electrophoresis (PAGE) Amany Elsayed
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.
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. There are several key steps: extraction of proteins from a sample, separation by size using gel electrophoresis, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, incubation with primary and secondary antibodies to detect the target protein, and use of a substrate to visualize the antibody-protein complex. Western blotting has applications in disease diagnosis, detecting defective proteins, and confirming the presence of viruses or bacteria.
Protein microarrays, ICAT, and HPLC protein purificationRaul Soto
The document discusses the Isotope-Coded Affinity Tag (ICAT) method for protein quantification and identification. ICAT uses chemical labeling reagents that specifically label cysteine residues. There are 4 main steps: 1) Lyse and label protein samples from two states with light and heavy ICAT tags, 2) Mix and proteolyze samples to generate peptide fragments, some tagged, 3) Isolate tagged fragments using avidin affinity chromatography, 4) Analyze isolated peptides using mass spectrometry to identify and quantify proteins between the two states. ICAT allows accurate quantification of complex protein mixtures.
This document discusses various biophysical and biochemical techniques used to analyze biotech products, including proteins. It describes techniques such as circular dichroism spectroscopy, fluorescence spectroscopy, calorimetry, sedimentation velocity, and western blotting that can provide information on a protein's secondary structure, tertiary structure, stability, and post-translational modifications. It also discusses analytical methods like chromatography, electrophoresis, mass spectrometry, and spectroscopy that can analyze a protein's purity, heterogeneity, conformation, and interactions with other molecules. The techniques allow characterization of biotech products to ensure quality, identity, stability and activity.
Western blotting is a laboratory technique used to detect specific proteins in a mixture. It works by separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. The key steps are sample preparation, gel electrophoresis to separate proteins, transferring proteins to a membrane, blocking the membrane to reduce background noise, incubating with primary antibodies that bind to the target protein, incubating with secondary antibodies linked to enzymes, and detecting the target protein through an enzymatic reaction. Western blotting is used to determine the size and amount of proteins and diagnose diseases by detecting antibodies.
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.
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.
Gel Based Proteomics and Protein Sequences AnalysisGelica F
Two-dimensional gel electrophoresis (2DE) is the standard method for quantitative proteome analysis. It combines protein separation based on isoelectric focusing and molecular weight. In the first dimension, proteins are separated based on their isoelectric point using immobilized pH gradients. In the second dimension, proteins are separated by molecular weight using SDS-PAGE. The separated protein spots are then analyzed using mass spectrometry to identify individual proteins. 2DE provides high resolution and the ability to analyze thousands of proteins simultaneously, but it also has limitations including irreproducibility and inability to resolve all proteins.
Analytical techniques for separation or purification of proteinsrohini sane
A comprehensive presentation on Analytical techniques for separation or purification of proteins for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.
2D-PAGE is a technique used to separate complex protein mixtures based on isoelectric point and molecular weight. It involves two sequential steps - isoelectric focusing and SDS-PAGE. In isoelectric focusing, proteins are separated based on their isoelectric point in an immobilized pH gradient. They are then separated by SDS-PAGE based on their molecular weight. The separated proteins can then be visualized through staining and identified through mass spectrometry. While useful for proteomic analysis, 2D-PAGE has limitations such as low reproducibility and dynamic range.
Electrophoresis is a technique used to separate charged biomolecules like proteins and nucleic acids. It works by applying an electric field to migrate these molecules through a buffer solution or gel based on their charge to mass ratio. The first major use of electrophoresis was in 1937. Key aspects include using a buffer to maintain pH and conductivity, as well as staining and destaining techniques to visualize separated biomolecules. Common types include gel electrophoresis using agarose or polyacrylamide gels as well as paper electrophoresis. Applications include clinical diagnosis and protein research.
Proteins – Basics you need to know for ProteomicsLionel Wolberger
The document provides an overview of key concepts in proteomics, including:
1) It discusses protein structure and function, the 20 common amino acids, and post-translational modifications that proteins undergo.
2) It introduces common techniques used in proteomics like chromatography, electrophoresis, mass spectrometry, and bioinformatics.
3) It summarizes protein analysis methods like gel electrophoresis, isoelectric focusing, and immunological assays used to detect and purify proteins of interest.
This document provides an overview of various protein purification techniques, including:
- Chromatography methods like affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography.
- Key steps in protein purification like cell lysis, centrifugation, assaying fractions for protein and activity, and monitoring purity with SDS-PAGE gels.
- Considerations for each chromatography method like matrix selection, binding capacities, and driving forces.
- Emerging techniques like capillary electrochromatography and multi-dimensional separations.
The document discusses proteomics, which is the study of the entire complement of proteins in a cell or organism. It defines key proteomics terms like proteome and describes techniques used in proteomics like protein separation, 2D gel electrophoresis, mass spectrometry, and protein digestion. The goals of proteomics include detecting and comparing protein expression profiles to understand biological processes and discover drug targets. Proteomics provides important insights not available through genomics alone.
Electrophoresis is a method used to separate macromolecules like DNA, RNA, and proteins based on their size and charge. There are several electrophoretic techniques including gel electrophoresis using agarose, polyacrylamide, or gradient gels, as well as capillary electrophoresis, isoelectric focusing, and microchip electrophoresis. Two-dimensional gel electrophoresis separates proteins in two steps based on isoelectric point and molecular weight to analyze complex protein mixtures.
This study used zymography to detect proteolytic enzymes in wheat leaves. One-dimensional (1D) zymography revealed 7 bands of proteolytic activity, which were assigned to proteinase families using specific inhibitors. Two-dimensional (2D) zymography provided additional separation of proteases according to isoelectric point, revealing multiple isoforms within families. Metalloproteinases were detected as a 150 kDa band inhibited by EDTA. Aspartic proteinases appeared as a 115-118 kDa double band inhibited by pepstatin. Serine and cysteine proteinases of various sizes were also observed. 2D zymography enabled visualization of isoforms within proteinase families.
The document discusses purification of recombinant proteins using affinity tags. It describes immobilized metal affinity chromatography (IMAC) as a widely used method to purify recombinant proteins fused to tags like histidine, GST or MBP. The document outlines the steps involved, including gene amplification, cloning, expression in bacteria or yeast, and purification. It focuses on using histidine tags and nickel-chelate affinity chromatography, noting the advantages of tags for simplifying purification and detection of recombinant proteins.
This document provides an overview of analytical proteomics and its applications. It defines the proteome and proteomics, and discusses the difference between protein biochemistry and proteomics. It describes the key steps in analytical proteomics: protein separation techniques like 1D/2D gel electrophoresis and isoelectric focusing; protein digestion using proteases like trypsin; and protein identification via mass spectrometry. Finally, it outlines some applications of proteomics like comparing proteomes under different conditions to study biological processes.
The document discusses aptamers, which are oligonucleotides or peptides that bind to targets with high affinity and specificity due to their unique three-dimensional structures. It describes how aptamers are produced through an in vitro selection process called SELEX and classified based on their structure and selection technique. The document also compares aptamers to antibodies and outlines several applications of aptamers in therapeutics, drug delivery, bioimaging, diagnostics, and more.
Scott Malcolm | Describe About Purpose and Creation Process of Gel Electropho...Scott Malcolm Dallas
Scott Malcolm is a great businessman, who lives in Dallas, Taxes. He explains here about biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length and to estimate the size of DNA and RNA fragments.
This document summarizes techniques for exploring and analyzing proteins, including concentrating purified proteins using lyophilization or ultrafiltration, separating proteins using electrophoresis or mass spectrometry, and identifying proteins using mass spectrometry. Electrophoresis techniques like SDS-PAGE and 2D gels separate proteins based on size and charge, allowing visualization and quantification of purified proteins. Mass spectrometry further identifies proteins by correlating detected ion masses with known protein standards. These techniques provide a quantitative evaluation of protein purification schemes.
Polyacrylamide gel electrophoresis (PAGE) Amany Elsayed
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.
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. There are several key steps: extraction of proteins from a sample, separation by size using gel electrophoresis, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, incubation with primary and secondary antibodies to detect the target protein, and use of a substrate to visualize the antibody-protein complex. Western blotting has applications in disease diagnosis, detecting defective proteins, and confirming the presence of viruses or bacteria.
Protein microarrays, ICAT, and HPLC protein purificationRaul Soto
The document discusses the Isotope-Coded Affinity Tag (ICAT) method for protein quantification and identification. ICAT uses chemical labeling reagents that specifically label cysteine residues. There are 4 main steps: 1) Lyse and label protein samples from two states with light and heavy ICAT tags, 2) Mix and proteolyze samples to generate peptide fragments, some tagged, 3) Isolate tagged fragments using avidin affinity chromatography, 4) Analyze isolated peptides using mass spectrometry to identify and quantify proteins between the two states. ICAT allows accurate quantification of complex protein mixtures.
This document discusses various biophysical and biochemical techniques used to analyze biotech products, including proteins. It describes techniques such as circular dichroism spectroscopy, fluorescence spectroscopy, calorimetry, sedimentation velocity, and western blotting that can provide information on a protein's secondary structure, tertiary structure, stability, and post-translational modifications. It also discusses analytical methods like chromatography, electrophoresis, mass spectrometry, and spectroscopy that can analyze a protein's purity, heterogeneity, conformation, and interactions with other molecules. The techniques allow characterization of biotech products to ensure quality, identity, stability and activity.
Western blotting is a laboratory technique used to detect specific proteins in a mixture. It works by separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein. The key steps are sample preparation, gel electrophoresis to separate proteins, transferring proteins to a membrane, blocking the membrane to reduce background noise, incubating with primary antibodies that bind to the target protein, incubating with secondary antibodies linked to enzymes, and detecting the target protein through an enzymatic reaction. Western blotting is used to determine the size and amount of proteins and diagnose diseases by detecting antibodies.
Western blotting by Shahzad Naseer AwanShahzad Awan
The document describes the process of Western blotting, which is used to detect specific proteins in a tissue sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to detect the protein of interest. The key steps are tissue preparation, gel electrophoresis to separate proteins, transfer to a membrane, blocking, primary/secondary antibody incubation, and detection of the protein-antibody complex. Western blotting allows detection of a specific protein from a mixture and estimation of its molecular weight.
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
Methods in Protein Biochemistry BY
GLOBALWEBTUTORS.COM. get instant help for your Biochemistry assignment from globalwebtutors.com where highly skilled professionals are always available for helping you
Western blot is a commonly used method for protein analysis. It can be used for qualitative and semi-quantitative protein analysis. For the accomplishment of the western blot, there are three elements, separation of proteins by size, transferring proteins to a solid support, and marking proteins by primary and secondary antibodies for visualization.
Western blotting is a technique used to detect specific proteins in a sample. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a target protein based on its molecular weight and signal intensity. Key steps include sample preparation, electrophoresis to separate proteins by size, electrotransfer to a membrane, blocking to reduce background noise, probing with primary and secondary antibodies, washing, and detection of the target protein. The technique allows identification and quantification of proteins but has limitations related to its qualitative nature and specificity of antibodies used.
The document provides a detailed overview of the Western blot technique. It describes the key steps as:
1) Tissue preparation and protein extraction from samples.
2) Separation of proteins by gel electrophoresis based on molecular weight.
3) Transfer of proteins from the gel to a membrane for detection.
4) Blocking of the membrane to prevent non-specific antibody binding.
5) Detection of target proteins using primary and secondary antibodies, and various detection methods.
Protein purification involves a series of steps to isolate a single protein from a complex mixture. These steps may separate proteins based on size, charge, or binding affinity. Common techniques include precipitation with ammonium sulfate, chromatography methods like ion exchange, affinity, size exclusion, and reversed-phase chromatography, and electrophoresis. The goal is to free the protein of interest from other materials, separate it from other proteins, and finally isolate it in a pure form for characterization and use.
This document discusses protein folding and misfolding. It begins by introducing how the three-dimensional structure of proteins, which is key to their function, is acquired through folding of the polypeptide chain. Changes to the chain through gene variations or modifications can alter the folding process and cause misfolding. The document then discusses how protein misfolding diseases can result from changes in cellular environment or overload of the protein quality control system. It provides examples of protein quality control systems and chaperones that assist folding, prevent aggregation, and eliminate misfolded proteins. The concept of a protein folding energy landscape is also introduced to explain how proteins search for their stable native state.
Introduction and Description to Western Blotting, Steps involved in Western Blotting- Sample Preparation, Protein Gel Electrophoresis, SDS-PAGE, Protein Transfer, Electrophoretic Protein Transfer, Transfer Sandwich Diagram, Blocking, Antibody Probing and Detection, Applications of Western Blotting.
Western blotting is a technique used to detect specific proteins in a complex protein mixture. It involves transferring proteins separated by gel electrophoresis onto a membrane and using antibodies to identify a target protein. The key steps are sample preparation, gel electrophoresis to separate proteins, protein transfer to a membrane, blocking to prevent nonspecific antibody binding, primary and secondary antibody probing, and detection of the target protein with an enzyme substrate reaction. Western blotting is widely used to detect viral proteins, characterize antibodies, and study the immune response.
This document discusses various techniques used in immunoblotting and blotting. It begins by defining blotting as techniques used to visualize specific DNA, RNA, and proteins among contaminants. It then describes three main types of blotting - western blotting for proteins, northern blotting for RNA, and southern blotting for DNA. The document focuses on western blotting and immunoblotting. It provides details on tissue preparation, gel electrophoresis, protein transfer, blocking, detection, analysis, and applications of western blotting and immunoblotting techniques.
Protein purification involves isolating a protein of interest from a complex mixture through a series of separation processes. These processes exploit differences in protein properties like size, charge, and binding affinity. Key steps include cell lysis, precipitation, centrifugation, chromatography, and electrophoresis to separate the target protein from others in the sample. The purified protein can then be characterized and used for various analytical and preparative purposes.
Immobilization of proteins on the solid support of nitrocellulose membrane or polyvinylidinefluoride membrane. Then antibodies bind speciffcally that can be analyzed through Autoradiography
Inclusion bodies are aggregates of proteins that form when proteins are overexpressed in cells. They typically contain high levels of the overexpressed protein with little other cellular components. While inclusion bodies were traditionally thought to only contain misfolded proteins, some evidence indicates proteins in inclusion bodies can retain native structure. Several methods exist for recovering active proteins from inclusion bodies, including dilution, chromatography, and adding compounds to aid refolding. Fusion tags are often used to improve expression and purification of recombinant proteins by enhancing solubility, aiding detection and purification, and more. Enzymatic cleavage is commonly used to remove fusion tags after purification.
Protein purification techniques take advantage of differences in protein properties like charge, size, and binding affinity. The first step is breaking open cells to release proteins. Centrifugation is used to separate subcellular components. Fractionation utilizes differences in solubility, often using ammonium sulfate precipitation. Dialysis removes small solutes by exchanging them through a semipermeable membrane. Column chromatography separates proteins as they migrate through a solid matrix at different rates depending on their interactions. Specific techniques further separate proteins based on ionic charge using ion-exchange, size using size exclusion, or binding affinity using affinity chromatography.
This document discusses several methods for isolating and separating proteins, including:
- Ultracentrifugation, which separates proteins based on mass and density over time in a centrifuge.
- Dialysis, which removes smaller molecules from a protein solution through a semi-permeable membrane.
- Chromatography techniques like gel filtration, ion exchange, and affinity chromatography separate proteins based on size, charge, and binding affinity to separate mixtures.
- Electrophoresis techniques like agarose/polyacrylamide gel electrophoresis and 2D electrophoresis separate proteins based on size and isoelectric point to further purify samples.
Proteomics is the study of the complete set of proteins expressed in an organism under particular conditions. It aims to understand protein expression in response to changing conditions like disease. Tools in proteomics include cell lysis, fractionation, protein concentration and quantification, digestion, and peptide cleanup prior to mass spectrometry analysis. Key techniques discussed are molecular techniques like SAGE, separation techniques like gel electrophoresis and chromatography, and protein identification techniques like mass spectrometry.
Conjugation is the method of adding an antigen to a larger molecule that ensures that the antigen stimulates the immune response that generates antibodies.
This guide is key to successful IHC experiments. Since no universal tissue preparation method will be ideal for all sample and tissue types, all protocols given here are intended as a starting point from which the experimenter must optimize as needed.
Immunohistochemistry (IHC) is a method that combines biochemical, histological and immunological techniques into a simple but powerful assay for protein detection. IHC provides valuable information as it visualizes the distribution and localization of specific cellular components within cells and in proper tissue context.
Fa cs sample preparation & protocolJames Waita
Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different subpopulations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell.
Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different sub- populations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell.
Western blotting protocol & troubleshootingJames Waita
This document provides a recommended protocol for performing a Western Blot, including procedures for protein extraction from cells and tissues, protein quantification, gel electrophoresis, protein transfer to a membrane, membrane blocking, and antibody incubation. The key steps are extracting proteins from samples using lysis buffers, quantifying protein concentration using a BCA assay, separating proteins by SDS-PAGE gel electrophoresis, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, and detecting target proteins by incubating the membrane with primary and secondary antibodies. Troubleshooting tips are also provided throughout the protocol.
ELISA troubleshooting guide serves as a checklist for the possible causes and solutions with respect to some of the most commonly encountered problems from the ELISA assays.
Describing and Interpreting an Immersive Learning Case with the Immersion Cub...Leonel Morgado
Current descriptions of immersive learning cases are often difficult or impossible to compare. This is due to a myriad of different options on what details to include, which aspects are relevant, and on the descriptive approaches employed. Also, these aspects often combine very specific details with more general guidelines or indicate intents and rationales without clarifying their implementation. In this paper we provide a method to describe immersive learning cases that is structured to enable comparisons, yet flexible enough to allow researchers and practitioners to decide which aspects to include. This method leverages a taxonomy that classifies educational aspects at three levels (uses, practices, and strategies) and then utilizes two frameworks, the Immersive Learning Brain and the Immersion Cube, to enable a structured description and interpretation of immersive learning cases. The method is then demonstrated on a published immersive learning case on training for wind turbine maintenance using virtual reality. Applying the method results in a structured artifact, the Immersive Learning Case Sheet, that tags the case with its proximal uses, practices, and strategies, and refines the free text case description to ensure that matching details are included. This contribution is thus a case description method in support of future comparative research of immersive learning cases. We then discuss how the resulting description and interpretation can be leveraged to change immersion learning cases, by enriching them (considering low-effort changes or additions) or innovating (exploring more challenging avenues of transformation). The method holds significant promise to support better-grounded research in immersive learning.
When I was asked to give a companion lecture in support of ‘The Philosophy of Science’ (https://shorturl.at/4pUXz) I decided not to walk through the detail of the many methodologies in order of use. Instead, I chose to employ a long standing, and ongoing, scientific development as an exemplar. And so, I chose the ever evolving story of Thermodynamics as a scientific investigation at its best.
Conducted over a period of >200 years, Thermodynamics R&D, and application, benefitted from the highest levels of professionalism, collaboration, and technical thoroughness. New layers of application, methodology, and practice were made possible by the progressive advance of technology. In turn, this has seen measurement and modelling accuracy continually improved at a micro and macro level.
Perhaps most importantly, Thermodynamics rapidly became a primary tool in the advance of applied science/engineering/technology, spanning micro-tech, to aerospace and cosmology. I can think of no better a story to illustrate the breadth of scientific methodologies and applications at their best.
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
The binding of cosmological structures by massless topological defectsSérgio Sacani
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
Travis Hills of Minnesota developed a method to convert waste into high-value dry fertilizer, significantly enriching soil quality. By providing farmers with a valuable resource derived from waste, Travis Hills helps enhance farm profitability while promoting environmental stewardship. Travis Hills' sustainable practices lead to cost savings and increased revenue for farmers by improving resource efficiency and reducing waste.
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
Phenomics assisted breeding in crop improvementIshaGoswami9
As the population is increasing and will reach about 9 billion upto 2050. Also due to climate change, it is difficult to meet the food requirement of such a large population. Facing the challenges presented by resource shortages, climate
change, and increasing global population, crop yield and quality need to be improved in a sustainable way over the coming decades. Genetic improvement by breeding is the best way to increase crop productivity. With the rapid progression of functional
genomics, an increasing number of crop genomes have been sequenced and dozens of genes influencing key agronomic traits have been identified. However, current genome sequence information has not been adequately exploited for understanding
the complex characteristics of multiple gene, owing to a lack of crop phenotypic data. Efficient, automatic, and accurate technologies and platforms that can capture phenotypic data that can
be linked to genomics information for crop improvement at all growth stages have become as important as genotyping. Thus,
high-throughput phenotyping has become the major bottleneck restricting crop breeding. Plant phenomics has been defined as the high-throughput, accurate acquisition and analysis of multi-dimensional phenotypes
during crop growing stages at the organism level, including the cell, tissue, organ, individual plant, plot, and field levels. With the rapid development of novel sensors, imaging technology,
and analysis methods, numerous infrastructure platforms have been developed for phenotyping.
2. Introduction
Principle
1. Protein Extraction and Quantitation
To extract protein for the western blot analysis, the first step is to prepare for the lysate from samples. Cell cultures and tissue
are the most common samples to start with. When preparing samples for Western blot it is useful to keep samples on ice until
the denaturation step. It may also be advantageous to add protease inhibitors and/or phosphatase inhibitors to prevent
sample degradation. A good example is p53 which is a phosphorylated protein that is highly sensitive to proteases. Without
precautions the protein and phosphorylation sites are destroyed leadingto false negative results.
Western blotting WB (immunoblotting) is a widely practiced analytical
technique to detect target proteins within samples using antigen-specific
antibodies. WB involves two major processes: Separation of soluble
proteins into discrete bands and transfer of those proteins onto a solid
matrix for subsequent analysis by immunological probes.
Under standard denaturing conditions, SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) is used to separate sample proteins based on
polypeptide length. The separated proteins are transferred (blotted) onto a
membrane matrix of nitrocellulose or PVDF where they are stained with
antibody probes specific to target protein antigens. Typically, blots are
incubated with antibodies against the antigen of interest followed by a
secondary antibody and detection.
Analysis of protein migration and the intensity of chromogenic,
chemiluminescent or fluorescent signals offer protein expression details
from cells or tissue homogenates. When coupled with high-resolution gel
electrophoresis, antibodies of strong avidity and specificity to target
antigens, and robust signal reporting, Western blotting can detect picogram
quantities of protein
2
3. Afterprotein extraction, the concentrationof protein should be measured to ensure that equalamount is loaded onto the
gel.At Boster, we have developed three protein assaykits for this purpose:
The Coomassie Plus Protein Assay is easy to carry out and consumes less sample. However, there can be considerable
variability among proteins in this assay that impacts linearity. The pH values may also change as a function of a variety of
elements such as high concentration of Tris, EDTA, urea, glycerol, sucrose, acetone, ammonium sulfate and detergent.
Further, the pH change willsubsequently influence color development and interfere with protein quantification.
The BCA protein assay is valued for increased stability, higher sensitivity and greater flexibility. The colored complex formed
is more stable with few interfering substances. Therefore, this assay has an obvious advantage over the Coomassie Plus
Protein Assaywhich is influenced by detergent and pH.
2. Protein Separation: Electrophoresis
Electrophoretic separation describes the phenomenon of charged particles moving through a gel matrix under the influence
of an electric field. It is used to separate proteins according to their electrophoretic mobility depending on the charge, size
and structure of the molecules. Polyacrylamide gels (PAGE) are three-dimensional mesh polymers composed of cross-linked
acrylamide. As a supporting matrix PAGE gels have several desirable features that make it a versatile medium. It is thermo-
stable, transparent, strong, and can be prepared with a wide range of average pore sizes.
Denatured Proteins
Denatured proteins are most commonly used for Western blotting. Native protein structures may
include inter- and/or intra-chain bonds creating secondary or tertiary structures which may play a
vital role in the activityof
the protein. However, these structures also affect the migration of the protein through the pores created by the
cross-linked polyacrylamide.For example, a low molecular weight protein with a complex tertiary structure may
migrate at the same rate as a largerprotein with a simpler structure indicating an apparently similarmolecular
weight. It is important to note that many antibodies are generated against peptides and often better recognize
protein in the denatured state. Denaturation of proteins is accomplished in three ways:
BCA (Bicinchoninic Acid) Protein Assay Kit (Boster Catalog# AR0146) Micro BCA
(Bicinchoninic Acid) Protein AssayKit (Boster Catalog# AR1110) Coomassie Plus Protein
AssayKit (Boster Catalog# AR0145)
3
4. linearized proteins that isproportional to its peptide length. In the presence of SDS, electrophoretic mobility
is largely based on molecule weight.
(ii) Strong reducing agents such as β-mercaptoethanol (BME) and dithiothreitol (DTT) disrupt disulfide linkages
between cysteine residues within or between protein chains.
(iii)Heating the samples to 100°C further promotes protein denaturation and SDS binding, rod- shape formation,
and negativecharge adherence.
Loading buffers containing tracking dyes such as bromophenol blue dye may be added to the protein solution to
follow the progress of the proteins through the gel during the electrophoretic run.
“Native” orNon-Denatured Proteins
Separation of non-denatured proteins relies on the sum of intrinsic charges carried on a protein and is
accomplished through isoelectric focusing (IEF). IEF employs polyacrylamidegels designed to contain a continuum
of pH zones where a protein stops migrating under the influence of electric current where it has no net charge, its
isoelectric point (pI). This pI may be variablefor a given protein and indicative of the presenceof post-translational
modification such as sugars(glycosylation) or phosphates (phosphorylation). IEF separation may be accomplished by
the addition of isophores to a polyacrylamidegel preparation prior to crosslinking (casting gels), immobilization of
pH gradients to a solidsupport (IPG strips), or using
a solution phase technique.
Gel Percentage and Pore Size
The use of (PAGE) gels for separating proteins ranging in size from 5-2,000 kDa is made possible by the
uniform pore size created during polymerization of acrylamide. Pore size is controlled via the ratios of
acrylamide to bis- acrylamide in gel making process. Typical PAGE gels are cast with a stacking gel (5%)
poured over the top of
a resolving gel. The relatively low concentration stacking layer is designed to counteract the migration differential of protein
samples as they first enter the separation matrix. At the interface of the two gel components, the proteins are “stacked” to
optimize their separation during their migration through the resolving gel. Resolving gels are made in 5%, 8%, 10%, 12%
or 20% formulations, each providing a range of protein separation based on apparent molecular weight. Gel
4
5. Buffer Systems
A widevarietyof buffer systems existfor use with PAGE. The most well-known isthe tris-glycine or "Laemmli"
discontinuous buffer system which uses a stackinggel prepared with buffer of pH
6.8 and a resolving gel at a pH of ~8.3-9.0. Drawbacks associated with this system include: potential protein deamination
and alkylationor disulfide bond formation between cysteine residues
from the re-oxidation of cysteine due to non-migration of sample buffer reducing agents into the resolving gel.
Additionally, different buffers are required for the cathode and anode ends of the
gel. Evolutions in buffering technology (e.g.,bis-Tris) avoid these pitfallsby casting and running gels under slightly
acidic (pH ~6.5) conditions and include reducing agents (e.g. sodium bisulfite)
that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers
with lower pH values is that the acrylamide gel is more stable at lower pH
values,so the gels can be stored for long periods of time before use.
As voltage is applied to a Tris-glycine system the anions (and negatively charged sample molecules) migrate toward the
positive electrode (anode) in the lower chamber. The leading anion is Cl-(high mobility and high concentration); glycinate
is the trailing ion (low mobility and low concentration). SDS-protein particles do not migrate freely at the border
between the Cl- of the gel buffer and the Gly- of the cathode buffer. Because of the voltage drop between the Cl-and
glycine buffers, proteins are compressed (stacked) into micrometer thin layer between the stacking and resolving gels. In
resolving gel, proteins with more negative charges per unit migrate faster than those with less negative charges per unit.
That is, proteins with small molecular weight migrate faster than proteins with large molecular weight. The boundary
moves through a pore gradient and the protein stack gradually disperses due to a frictional resistance increase of the gel
matrix. For every protein at a different position, stackingand un-stacking occur continuously in the gradient gel.
3. Protein Transfer
Transfer Types
Two main types of procedures and associated apparatus are used to perform electrophoretic transfer of
proteins. Each of them offers advantages and is selected based upon experimental requirements and lab
preferences. 5
6. short due to both the high electric field caused by the close proximity between the two electrodes and by the low
buffer capacity of the saturated filter paper. High field strengths may cause some small proteins to be driven
through the transfer membrane, while shortened run times may result in the inefficient transfer of some large
proteins. Semi-dry protein transfer methods are not considered to be reliablefor quantitative analysis.
(i)Wet Transfer
In a wet transfer process, the gel-membrane-filter sandwich is flanked by pads or sponges and placed vertically in a
cassette or transfer tank filled with buffer. As in the semi-dry transfer proteins migrate from the gel to the
membrane under the electric field produced by the parallel electrodes. Large buffer capacities with wet transfer
procedures offer more flexibility for voltage settings, transfer times, and temperature conditions. Varying these
parameters may allow some larger proteins to be transferred more effectively while promoting efficient binding of
lower molecular weight proteins. While wet transfer methods require more complex apparatus and higher buffer
costs, the opportunities for optimization of transfer conditions make the method a preferred choice when
quantitation of target proteins are desired.
Transfer Membrane
Transfer membranes are selected based on their physical properties that lend them for use with particular methods and
conditions. Protein binding capacity, mechanical strength, and downstream analysis such as re-probing or protein
sequencing are considerations when choosing a blotting membrane.
(i) Nitrocellulose
Nitrocellulose, one of the first materials used for transfer membranes, is still considered a good choice for general
purpose western blotting. Nitrocellulose is inexpensive, has a high affinity for proteins, is easily prepared for
blotting, and can be used with a variety of detection systems. Due to its brittle nature nitrocellulose is not a
preferred material for blots intended for workflows requiring repeated handling or manipulations. Some
manufacturers offer nitrocellulose membranes with a synthetic backing giving increased strength while retaining
the desirable characteristics of protein binding and ease of wetting. When used as a transfer membrane
Nitrocellulose requires the use of methanol in the transfer buffer to remove SDS bound to proteins, thus enabling
hydrophobic interaction of the protein with the transfer membrane. Methanol also has a shrinkage effect on the
6
7. (ii)Polyvinyl Difluoride (PVDF)
Membranes made of PVDF have some distinct advantages over nitrocellulose such as higher binding capacity (170-
200 µg/cm2 vs. 80-100 µg/cm2) and durability. The higher binding capacity is advantageous when attempting to
detect low expression proteins, but may also lead to higher background noise during detection. Prior to use, PDVF
membranes must be thoroughly wet in 100% methanol and then equilibrated in transfer buffer by soaking for at
least 5 minutes. Advantages of PVDF membranes are the opportunities for re-probing the transferred proteins after
a stripping procedure. WB Stripping Buffer (AR0153) can be used to remove bound primary and secondary
antibodies. Quantitative analysis of detected proteins on re-probed gels should be avoided as the stripping process
mayremove some targetproteins from the membrane.
Membrane Pore Size
Blotting membranes are available at typicalpore sizes of 0.2 µm, and 0.45 µm. For proteins of 20 kDa or smaller, 0.2
µm pore size is recommended, and 0.45 µm pore if your protein is greater than
20 kDa. If your application includes quantitation of your target protein, or your protein concentration is low, the
generalrule is to use the membrane with the smallerpore size.
Membrane Formats
(i) Pre-cut membranes are available for all of the most commonly used sizes of separation gels. Pre-cut and packaged
by manufacturers, these membranes may provide more consistent protein transfers than membranes cut from rolls
or largesheets.
(ii) Rolls of membranes are available for cutting membranes yourself in your laboratory to use with a variety of gelsizes.
The lower cost of the membrane may be offset by the time expended to ensure the size of the transfer membrane
exactlymatches that of the separation gel to avoid introducing variabilityin transfer analysis.
4. MembraneBlocking
Following protein transfer, it is important to block the unreacted sites on the membrane using inert proteins and/or nonionic
detergent to reduce levels of nonspecific protein binding during the assay. Blocking buffers should block all unreacted sites
7
8. phosphorylated proteins. BSA or casein in TBS are recommended for phosphorylated target analysis or when using alkaline
phosphatase-based detection methods.
Note: Use of NaN3 as a preservative in blocking reagents should be avoided when using peroxidase detection systems due
to its oxidase inhibiting properties.
5. PrimaryAntibody Incubation
After blocking the transfer membrane, a primary antibody specific to a binding site on the target protein is
diluted as appropriate and incubated with the membrane. This procedure is typically carried out in a dish or
tray of dimensions allowing the membrane
to layhorizontal whileimmersed in buffer. Incubation is performed overnight at 4ºC or 1-2 hours at RT with mild agitation.
The choice of a primary antibody depends on the antigen to be detected. Both polyclonal and monoclonal antibodies work
well for western blot analysis. Monoclonal antibodies recognize single specific antigenic epitope. The higher specificity may
result in lower background on analysis. Target epitopes that are blocked or destroyed may result in poor blot results when
monoclonal antibodies are used as probes. Polyclonal antibodies may recognize more epitopes on the target and they often
have higher affinity providing results for analysis when specific epitopes are notknown or their identification is not required
for the application. Polyclonalantibodies are generally found at lower cost than monoclonal antibodies.
6. Secondary Antibody Incubation
After washing the membrane to remove unbound primary antibody, the membrane is exposed to an enzyme
conjugated secondary antibody. The secondary antibody is highly specific for a site on the primary antibody
and is engineered bind tightly. The most
popular secondary antibodies are anti-mouse and anti-rabbit immunoglobulins (Ig) as these animals are
commonly used as host species for production of primary antibodies. In turn, goats are used to raise anti-mouse and anti-
rabbit polyclonal antibodies. Thus, goat anti-mouse and goat anti-rabbit immunoglobulins are the most commonly used
secondary antibodies. The choice of secondary antibody depends upon the species of animal in which the primary antibody
was raised. For example, if the primary antibody is a mouse monoclonal antibody, the secondary antibody must be anti-
mouse. If the primary antibody is a rabbit polyclonal antibody, the secondary antibody must be anti-rabbit. The secondary
antibody must also recognize the isotype of the primary antibody (IgG1, IgM, etc.).8
9. Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes that are used extensively. As a
result of alkaline phosphatase (AP) catalysis, the colorless substrate BCIP will be converted to a blue product. In the
presence of H2O2, 3-amino-9- ethyl carbazole and 4-chlorine naphthol will be oxidized into brown and blue products,
respectively. The figure below shows two WBresults by using Boster’sDAB chromogenic detection system.
TissueLysates
Lane 1:Rat skeletalmuscle Lane 2:Rat liver
Lane 3:Rat brain
Lane 4:Rat small intestine Whole Cell Lysates
Lane 5:MCF-7
Lane 6:HeLa
Lane 7:Smmc
Lane 8:Jurkat
Lane 9:Colo320
Tissue Lysates Lane 1: Rat brain Lane 2:Rat
brain Lane 3:Rat liver Lane 4:Rat pancreas
Whole Cell Lysate Lane 5:MCF-7
Chemiluminescence
Enhanced chemiluminescence is another method that employs HRP detection. Using HRP as the enzyme label, the
luminescent substrate (Luminol) will be oxidized by H2O2 and will produce light (luminesce). Luminiscence enhancers
combined with this substrate will induce a 1000-fold increase in light intensity. The light is then detected and captured
on photographic film. Film can be exposed for multiple time points to optimize the signal to noise ratio. The figure on
the next page shows three WBresults by using Boster’s ECL detection system.
9
10. Whole Cell Lysates Whole Cell Lysates Tissue Lysates
Lane 1:MCF7 Lane 1:MM453 Lane 1:Rat Ovary
Lane 2:Hela Lane 2:MM231 Lane 2:Human placenta
Lane 3:Jurkat Lane 3:Hela
Lane 4:HT1080 Lane 4:HT1080
Lane 5:Colo320 Lane 5:Colo320
Fluorescence
Fluorescence detection uses fluorochrome conjugated primary or secondary antibodies to detect the protein of interest.
Data is captured using specialized fluorescent imaging systems. There are several advantages to this method including the
elimination of the enzymatic step and the ability to multiplex. This method is also more quantitative than other
methods.
The choice of WB signal detection methods depends on the enzyme or detection agent bound to the secondary antibody.
ECL (Enhanced Chemiluminescent) and DAB chromogenic detection systems are typically used. In general, chromogenic
detection is not as popular as chemiluminescent due to its decreased sensitivity.
8. Controls
Proper control design is essential to successful western blot analysis. It will guarantee accurate and specific test results
identifying various problems quickly and precisely.We willdiscuss five control types as follows:
Positive Control
A lysate from a cell line or tissue sample known to express your target protein. Positive controls are designed to verify
working efficiencyof the antibodies.
10
11. Negative Control
A lysate from a cell line or tissue sample known not to express the protein you are detecting. Negative controls are
used is to check antibody specificity. Nonspecific binding and false positive result may be identified using negative
controls.
Secondary Antibody Control (No PrimaryAntibody Control)
A secondary antibody is incubated with the blot to check antibody specificity. Signals produced by nonspecific binding
may generate false positive results using the secondary alone.
Blank Control
Primary and secondary antibodies are not incubated with the membrane. Thisanalysis is performed to verify the transfer
membrane itselfdoes not induce a falsepositive result and is also used to check membrane blocking efficiency.
Loading Control
Loading controls are used to check sample quality and the performance of the secondary antibody
system. Loading controls are antibodies to "house-keeping proteins" or proteins that are expressed at equivalent levels
in almost all tissues and cells. Loading controls are required to check that equivalent quantities of target protein have
been loaded across gel lanes, especially when a comparison must be made between the expression levels of a protein in
different samples. They are also useful to check for effective transfer from the gel to the membrane across the entire
gel. Where uneven quantities of sample are loaded or even transfers have not occurred, the loading control bands can
be used to quantify the protein amounts in each lane. For publication-quality work, use of a loading control is
absolutely essential. Come commonly used loading controls are:
LoadingControl Molecular Weight (kDa) SampleType
Beta-Actin 43 WholeCell/cytoplasmic
GAPDH 30-40 WholeCell/cytoplasmic
Tubulin 55 WholeCell/cytoplasmic
VCDA1/Porin 31 Mitochondrial
COXIV 16 Mitochondrial
Lamin B1 16 Nuclear (Unsuitable for samples where nuclear envelope is removed)
TBP 38 Nuclear (Not suitable for samples whereDNA is removed)
11
12. WBResult by Beta-Actin Loading Control and Enhanced
Chemiluminescent (ECL) Substrate from Boster
WBResult by GAPDH Loading Control and Enhanced Chemiluminescent (ECL)
Substrate from Boster
12