BLOTTING
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Western blotting is a technique used to detect specific proteins in a sample containing a mixture of proteins. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a specific protein target through immunodetection. Key steps include extracting proteins, separating by SDS-PAGE gel electrophoresis, transferring to a membrane, blocking the membrane, incubating with primary and secondary antibodies, and detecting the target protein band using an enzyme substrate reaction. It is a widely used analytical technique in cell and molecular biology research.
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Western blotting
The document discusses Western blotting, which is a technique used to detect specific proteins in a biological sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are separating proteins by SDS-PAGE, transferring them to a membrane using blotting, and detecting the target protein with primary and secondary antibodies to visualize protein bands. Western blotting allows identification and quantification of proteins and is widely used in research and clinical diagnosis.
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DNA sequencing determines the order of bases in a DNA sample. Two major early methods are the Maxam-Gilbert chemical cleavage method using double-stranded DNA and the Sanger chain termination method using single-stranded DNA. The Sanger method involves primer annealing, complementary strand synthesis using labeled chain terminators, and resolution on a polyacrylamide gel to read the sequence. Bacteria protect their DNA from foreign DNA through modification and restriction. Modification involves host-specific methylation while restriction enzymes cut foreign DNA at specific recognition sequences. There are four classes of restriction enzymes that differ in subunit composition, cofactor requirements, and cleavage position relative to the recognition site.
Western Blotting Practical
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Blotting
The document describes various blotting techniques used to detect DNA, RNA, and proteins. It discusses Southern blotting, which is used to detect specific DNA sequences through separation, transfer, and hybridization. The overall steps involve DNA fragmentation, gel electrophoresis, transfer to a membrane, hybridization with a probe, washing, and detection. Northern blotting is similar but detects RNA and uses formaldehyde during gel electrophoresis. Western blotting detects specific proteins by separating proteins by size on a gel, transferring them to a membrane, labeling with antibodies, washing, and detecting the bound antibodies. These techniques allow for detection of specific biomolecules.
Western blotting pppt
Western blotting is a technique used to detect specific proteins in a sample based on antibody-protein binding. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a targeted protein. The process includes denaturing proteins, separating them by SDS-PAGE gel electrophoresis based on size, transferring to a membrane, blocking non-specific binding sites, incubating with primary and secondary antibodies, and detecting the targeted protein.
Western blotting
Western blotting is a technique used to detect specific proteins in a tissue sample. It involves separating proteins by gel electrophoresis based on size, transferring them to a membrane, and using antibodies to identify a target protein. The process starts with preparing the tissue sample, separating proteins by SDS-PAGE gel electrophoresis, transferring proteins to a membrane, blocking the membrane to reduce nonspecific antibody binding, probing with primary and secondary antibodies, washing unbound antibodies, and detecting the target protein. Western blotting is useful for applications like diagnosing diseases and studying gene expression.
Blotting techniques in Biotechnology
This document provides an overview of blotting techniques, including transfer methods, membranes used, and applications of Southern blotting, Northern blotting, and Western blotting. It discusses how blotting involves transferring proteins, DNA, or RNA from a gel onto a membrane. It describes the capillary, electro-blotting, and vacuum blot transfer processes and membranes like nitrocellulose, nylon, and PVDF. It then explains the specific methods and applications of Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins.
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Blotting techniques himanshu
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Western blotting
The document discusses Western blotting, which is a technique used to detect specific proteins in a biological sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are separating proteins by SDS-PAGE, transferring them to a membrane using blotting, and detecting the target protein with primary and secondary antibodies to visualize protein bands. Western blotting allows identification and quantification of proteins and is widely used in research and clinical diagnosis.
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DNA sequencing determines the order of bases in a DNA sample. Two major early methods are the Maxam-Gilbert chemical cleavage method using double-stranded DNA and the Sanger chain termination method using single-stranded DNA. The Sanger method involves primer annealing, complementary strand synthesis using labeled chain terminators, and resolution on a polyacrylamide gel to read the sequence. Bacteria protect their DNA from foreign DNA through modification and restriction. Modification involves host-specific methylation while restriction enzymes cut foreign DNA at specific recognition sequences. There are four classes of restriction enzymes that differ in subunit composition, cofactor requirements, and cleavage position relative to the recognition site.
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The document describes various blotting techniques used to detect DNA, RNA, and proteins. It discusses Southern blotting, which is used to detect specific DNA sequences through separation, transfer, and hybridization. The overall steps involve DNA fragmentation, gel electrophoresis, transfer to a membrane, hybridization with a probe, washing, and detection. Northern blotting is similar but detects RNA and uses formaldehyde during gel electrophoresis. Western blotting detects specific proteins by separating proteins by size on a gel, transferring them to a membrane, labeling with antibodies, washing, and detecting the bound antibodies. These techniques allow for detection of specific biomolecules.
Western blotting pppt
Western blotting is a technique used to detect specific proteins in a sample based on antibody-protein binding. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify a targeted protein. The process includes denaturing proteins, separating them by SDS-PAGE gel electrophoresis based on size, transferring to a membrane, blocking non-specific binding sites, incubating with primary and secondary antibodies, and detecting the targeted protein.
Western blotting
Western blotting is a technique used to detect specific proteins in a tissue sample. It involves separating proteins by gel electrophoresis based on size, transferring them to a membrane, and using antibodies to identify a target protein. The process starts with preparing the tissue sample, separating proteins by SDS-PAGE gel electrophoresis, transferring proteins to a membrane, blocking the membrane to reduce nonspecific antibody binding, probing with primary and secondary antibodies, washing unbound antibodies, and detecting the target protein. Western blotting is useful for applications like diagnosing diseases and studying gene expression.
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This document provides an overview of blotting techniques, including transfer methods, membranes used, and applications of Southern blotting, Northern blotting, and Western blotting. It discusses how blotting involves transferring proteins, DNA, or RNA from a gel onto a membrane. It describes the capillary, electro-blotting, and vacuum blot transfer processes and membranes like nitrocellulose, nylon, and PVDF. It then explains the specific methods and applications of Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins.
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.
Blotting techniques1
This document discusses different blotting techniques used to detect DNA, RNA, and proteins. Southern blotting is used to detect DNA and was developed by Edwin Southern in 1975. It involves separating DNA fragments by size, transferring them to a membrane, and using a probe to detect specific sequences. Northern blotting detects RNA and was developed in 1979. It similarly separates RNA and uses a probe to detect sequences of interest. Western blotting detects proteins and was developed in 1981. It separates proteins by SDS-PAGE gel electrophoresis, transfers them to a membrane, and uses an antibody probe to detect the target protein. These techniques are widely used in research and diagnostics.
Southern Blotting
Southern blotting is a technique used to detect specific DNA sequences. It involves digesting DNA with restriction enzymes, separating the fragments via electrophoresis, transferring the fragments to a membrane, and then detecting the fragments by hybridizing a probe. The key steps are digesting DNA, separating fragments, blotting to a membrane, hybridizing with a radioactive probe, and detecting fragments via autoradiography. Southern blotting is used for applications like diagnosing genetic diseases, paternity testing, and identifying infectious agents.
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This document discusses blotting techniques used in molecular biology. It describes how blotting involves transferring proteins, DNA, or RNA from a gel onto a membrane carrier using electrophoresis. The transferred molecules can then be visualized through staining, radioactive labeling, or by using specific probes like antibodies or hybridization probes attached to enzymes. The document outlines the basic three step process of blotting: 1) separating molecules by gel electrophoresis, 2) transferring molecules to a membrane, and 3) querying the membrane with probes to detect molecules of interest.
Blotting techniques
This document discusses various blotting techniques used to detect DNA, RNA, and proteins, including Southern blotting, Northern blotting, and Western blotting. It provides detailed descriptions of the procedures for each technique, including separating biomolecules by electrophoresis, transferring them to a membrane, hybridizing probes, and detecting bound probes. The techniques allow visualization and analysis of specific biomolecules within complex samples.
Blotting techniques
Southern blotting is a technique developed by Professor Edwin Southern in 1975 to detect specific DNA sequences. It involves separating DNA fragments by size, transferring them to a membrane, then using a probe to detect the bound fragment. It is used for gene mapping, evolution studies, and DNA fingerprinting. Northern blotting detects specific RNA sequences and was developed in 1979. RNA is separated by size and transferred to a membrane, then a probe detects bound RNA fragments. Western blotting detects specific proteins and was developed in 1981. Proteins are separated by gel electrophoresis, transferred to a membrane, and a primary antibody binds the target protein, which is then detected by a secondary antibody.
Blotting techniques
This document provides information on immunoblotting techniques such as Western blotting. It discusses the basic workflow of Western blotting which includes sample preparation, gel electrophoresis, protein transfer, blocking and antibody probing, detection, and analysis. It also summarizes other blotting techniques like Southern blot which is used to detect specific DNA sequences. The applications of Western and Southern blotting in research and clinical diagnosis are also briefly mentioned.
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Southern Blotting and Related DNA Detection Techniques
The document describes Southern blotting, a technique developed by Edwin Southern in 1975. It involves transferring DNA fragments separated by electrophoresis onto a membrane, where they can be detected through hybridization with labeled probes. Specifically, DNA is extracted, digested with restriction enzymes, separated on a gel, and transferred to a membrane via capillary action. The membrane-bound DNA can then be probed to detect specific fragments through hybridization and visualized. The Southern blot technique allows detection of a targeted DNA fragment against a complex background and has various applications in research, forensics, and medicine.
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What is blotting
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow researchers to detect specific DNA, RNA, and protein sequences by transferring them from a gel to a membrane and using probes to identify the targets. Southern blotting is used to detect DNA, Northern blotting detects RNA, and Western blotting identifies proteins. These techniques play important roles in research areas like gene expression analysis, disease diagnosis, forensics, and more by providing sensitive and specific detection of biomolecules.
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Southern blotting
Southern blotting is a technique used to identify specific DNA sequences within a mixture. It involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe to identify the target sequence through hybridization. Key steps include DNA extraction, restriction digestion, electrophoresis, depurination, denaturation, blotting, baking, hybridization, washing, and autoradiography. Southern blotting has applications in identifying viral/bacterial strains, studying mutations, forensic analysis, and mapping genomes. It allows for precise DNA identification.
Western blotting
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 locate the target protein. The key steps are sample preparation, SDS-PAGE gel electrophoresis to separate proteins by size, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, probing the membrane with antibodies to detect the target protein, washing unbound antibodies, and detecting the bound antibodies to analyze and image the results. Western blotting allows identification and quantification of proteins in a given sample.
Immunoblotting
This document provides an overview of immunoblotting techniques including ELISA, western blotting, and southern blotting. It discusses the basic principles, key steps, advantages, and applications of each technique. ELISA techniques include direct, indirect, sandwich, and competitive ELISA. Western blotting involves separating proteins by electrophoresis, transferring them to a membrane, and detecting specific proteins using antibodies. Southern blotting is used to detect specific DNA sequences and involves separating DNA fragments, transferring them to a membrane, and hybridizing a probe to the target sequence. Immunoblotting techniques are powerful tools for detecting and analyzing proteins or DNA in complex mixtures.
Blotting (Southern, Northern and Eastern)
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow for the transfer and detection of DNA, RNA, and proteins, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe for hybridization to detect specific DNA sequences. Northern blotting detects RNA transcripts by separating RNA on a gel, transferring it to a membrane, and labeling it with a probe. Western blotting identifies proteins by separating them on an SDS-PAGE gel, transferring them to a membrane, and using primary and secondary antibodies to detect specific proteins based on antigen-antibody binding. These techniques have various applications in research, diagnostics, and forensics
Southern Blotting Technique
This document describes the process of Southern blotting, which is used to detect specific DNA sequences in a sample. It involves purifying DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments by size via gel electrophoresis, transferring them to a membrane, then using a radioactive probe to detect fragments that hybridize based on complementary base pairing. The technique allows identification of mutations, deletions and other genetic variations, and has applications in medical diagnosis, forensics and other areas requiring DNA analysis.
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BLOTTING TECHNIQUES
This document discusses various blotting techniques used to identify specific DNA or RNA fragments, including Southern blotting for DNA, Northern blotting for RNA, and Dot blotting for DNA and RNA. It describes the process of Southern blotting, which involves digesting genomic DNA with restriction enzymes, separating fragments via gel electrophoresis, transferring DNA to a membrane, then using labeled cDNA probes to detect complementary DNA bands through autoradiography. Northern blotting follows a similar process to detect specific RNA molecules. These techniques allow for the identification and analysis of genes.
Westren and Northern blotting
Different blots are used to identify the presence of one specific target molecule (DNA, RNA or protein) in a complex mixture of related molecules. Blotting refers to the transfer of macromolecules (nucleic acids, proteins) from a gel onto the solid surface of an immobilized membrane for the detection of the transferred molecules.
Immunoblotting
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.
Western blottin-WPS Office.pptx
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.
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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.
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This document discusses different blotting techniques used to detect DNA, RNA, and proteins. Southern blotting is used to detect DNA and was developed by Edwin Southern in 1975. It involves separating DNA fragments by size, transferring them to a membrane, and using a probe to detect specific sequences. Northern blotting detects RNA and was developed in 1979. It similarly separates RNA and uses a probe to detect sequences of interest. Western blotting detects proteins and was developed in 1981. It separates proteins by SDS-PAGE gel electrophoresis, transfers them to a membrane, and uses an antibody probe to detect the target protein. These techniques are widely used in research and diagnostics.
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Southern blotting is a technique used to detect specific DNA sequences. It involves digesting DNA with restriction enzymes, separating the fragments via electrophoresis, transferring the fragments to a membrane, and then detecting the fragments by hybridizing a probe. The key steps are digesting DNA, separating fragments, blotting to a membrane, hybridizing with a radioactive probe, and detecting fragments via autoradiography. Southern blotting is used for applications like diagnosing genetic diseases, paternity testing, and identifying infectious agents.
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This document discusses blotting techniques used in molecular biology. It describes how blotting involves transferring proteins, DNA, or RNA from a gel onto a membrane carrier using electrophoresis. The transferred molecules can then be visualized through staining, radioactive labeling, or by using specific probes like antibodies or hybridization probes attached to enzymes. The document outlines the basic three step process of blotting: 1) separating molecules by gel electrophoresis, 2) transferring molecules to a membrane, and 3) querying the membrane with probes to detect molecules of interest.
Blotting techniques
This document discusses various blotting techniques used to detect DNA, RNA, and proteins, including Southern blotting, Northern blotting, and Western blotting. It provides detailed descriptions of the procedures for each technique, including separating biomolecules by electrophoresis, transferring them to a membrane, hybridizing probes, and detecting bound probes. The techniques allow visualization and analysis of specific biomolecules within complex samples.
Blotting techniques
Southern blotting is a technique developed by Professor Edwin Southern in 1975 to detect specific DNA sequences. It involves separating DNA fragments by size, transferring them to a membrane, then using a probe to detect the bound fragment. It is used for gene mapping, evolution studies, and DNA fingerprinting. Northern blotting detects specific RNA sequences and was developed in 1979. RNA is separated by size and transferred to a membrane, then a probe detects bound RNA fragments. Western blotting detects specific proteins and was developed in 1981. Proteins are separated by gel electrophoresis, transferred to a membrane, and a primary antibody binds the target protein, which is then detected by a secondary antibody.
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This document provides information on immunoblotting techniques such as Western blotting. It discusses the basic workflow of Western blotting which includes sample preparation, gel electrophoresis, protein transfer, blocking and antibody probing, detection, and analysis. It also summarizes other blotting techniques like Southern blot which is used to detect specific DNA sequences. The applications of Western and Southern blotting in research and clinical diagnosis are also briefly mentioned.
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This document provides an overview of various blotting techniques used to detect specific biomolecules, including Southern blotting, Western blotting, Northern blotting, and related methods. It describes the basic principles of blotting, which involve transferring biomolecules from a gel to a membrane for detection using probes or antibodies. Examples of applications for each technique are also outlined.
Southern Blotting and Related DNA Detection Techniques
The document describes Southern blotting, a technique developed by Edwin Southern in 1975. It involves transferring DNA fragments separated by electrophoresis onto a membrane, where they can be detected through hybridization with labeled probes. Specifically, DNA is extracted, digested with restriction enzymes, separated on a gel, and transferred to a membrane via capillary action. The membrane-bound DNA can then be probed to detect specific fragments through hybridization and visualized. The Southern blot technique allows detection of a targeted DNA fragment against a complex background and has various applications in research, forensics, and medicine.
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This document discusses various nucleic acid hybridization techniques. It begins with an introduction to DNA hybridization, including the principles of hybridization and basic procedures. It then describes several types of hybridization techniques: Southern hybridization detects DNA, Northern hybridization detects RNA, Western hybridization detects proteins, dot hybridization immobilizes fragmented DNA onto a membrane, and colony hybridization detects DNA in bacterial colonies. The document provides details on non-radioactive detection systems, the role of DNA probes, and comparing the techniques of Southern, Northern, and Western blotting.
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Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow researchers to detect specific DNA, RNA, and protein sequences by transferring them from a gel to a membrane and using probes to identify the targets. Southern blotting is used to detect DNA, Northern blotting detects RNA, and Western blotting identifies proteins. These techniques play important roles in research areas like gene expression analysis, disease diagnosis, forensics, and more by providing sensitive and specific detection of biomolecules.
Pulse Field Gel Electrophoresis
Pulsed-field gel electrophoresis (PFGE) is a technique used to separate large DNA molecules and generate DNA fingerprints for bacterial isolates. It involves using restriction enzymes to cut bacterial DNA into large fragments, which are then separated in an agarose gel using an electric field that periodically changes direction. PFGE allows discrimination of bacterial strains and is commonly used in epidemiological studies to link clinical infections to environmental or food isolates. While time-consuming, it provides stable, reproducible patterns and has been shown to be more discriminating than other subtyping methods for many bacteria.
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Southern blotting is a technique used to identify specific DNA sequences within a mixture. It involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, then using a labeled probe to identify the target sequence through hybridization. Key steps include DNA extraction, restriction digestion, electrophoresis, depurination, denaturation, blotting, baking, hybridization, washing, and autoradiography. Southern blotting has applications in identifying viral/bacterial strains, studying mutations, forensic analysis, and mapping genomes. It allows for precise DNA identification.
Western blotting
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 locate the target protein. The key steps are sample preparation, SDS-PAGE gel electrophoresis to separate proteins by size, transferring proteins from the gel to a membrane, blocking the membrane to prevent nonspecific antibody binding, probing the membrane with antibodies to detect the target protein, washing unbound antibodies, and detecting the bound antibodies to analyze and image the results. Western blotting allows identification and quantification of proteins in a given sample.
Immunoblotting
This document provides an overview of immunoblotting techniques including ELISA, western blotting, and southern blotting. It discusses the basic principles, key steps, advantages, and applications of each technique. ELISA techniques include direct, indirect, sandwich, and competitive ELISA. Western blotting involves separating proteins by electrophoresis, transferring them to a membrane, and detecting specific proteins using antibodies. Southern blotting is used to detect specific DNA sequences and involves separating DNA fragments, transferring them to a membrane, and hybridizing a probe to the target sequence. Immunoblotting techniques are powerful tools for detecting and analyzing proteins or DNA in complex mixtures.
Blotting (Southern, Northern and Eastern)
Blotting techniques such as Southern blotting, Northern blotting, and Western blotting allow for the transfer and detection of DNA, RNA, and proteins, respectively. Southern blotting involves separating DNA fragments via gel electrophoresis, transferring them to a membrane, and using a probe for hybridization to detect specific DNA sequences. Northern blotting detects RNA transcripts by separating RNA on a gel, transferring it to a membrane, and labeling it with a probe. Western blotting identifies proteins by separating them on an SDS-PAGE gel, transferring them to a membrane, and using primary and secondary antibodies to detect specific proteins based on antigen-antibody binding. These techniques have various applications in research, diagnostics, and forensics
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This document describes the process of Southern blotting, which is used to detect specific DNA sequences in a sample. It involves purifying DNA from cells, cutting the DNA into fragments using restriction enzymes, separating the fragments by size via gel electrophoresis, transferring them to a membrane, then using a radioactive probe to detect fragments that hybridize based on complementary base pairing. The technique allows identification of mutations, deletions and other genetic variations, and has applications in medical diagnosis, forensics and other areas requiring DNA analysis.
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BLOTTING TECHNIQUES
This document discusses various blotting techniques used to identify specific DNA or RNA fragments, including Southern blotting for DNA, Northern blotting for RNA, and Dot blotting for DNA and RNA. It describes the process of Southern blotting, which involves digesting genomic DNA with restriction enzymes, separating fragments via gel electrophoresis, transferring DNA to a membrane, then using labeled cDNA probes to detect complementary DNA bands through autoradiography. Northern blotting follows a similar process to detect specific RNA molecules. These techniques allow for the identification and analysis of genes.
Westren and Northern blotting
Different blots are used to identify the presence of one specific target molecule (DNA, RNA or protein) in a complex mixture of related molecules. Blotting refers to the transfer of macromolecules (nucleic acids, proteins) from a gel onto the solid surface of an immobilized membrane for the detection of the transferred molecules.
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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.
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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.
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BLO: Transferring the macromolecule from gel to membrane followed by detection on the membrane using antibody is k/a blotting
molecular methods used to identify and measure specific DNA, RNA and protein in complex biological mixtures.
It is the technique för
transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis.
IMMUNO BLOTTING:
Immunoblotting techniques use antibodies to identify target proteins .
They involve identification of protein target via antigen-antibody (or protein-ligand) specific reactions.
The Southern blot is used for transferring DNA,.
The Northern blot for RNA
The western blot for PROTEIN.
The Eastern blot for PROTEIN, post-translational modifications (PTMS) .
WESTERN BLOTTING:
Principle:
Western blotting technique is used for identification of particular protein from the mixture of protein.
In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test.
Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
METHODOLOGY:
Extraction of protein
2. Gel electrophoresis: SDS PAGE
3. Blotting: electrical or capillary blotting
4. Blocking: BSA
5. Treatment with primary antibody
6. Treatment with secondary antibody( enzyme labelled anti Ab)
7. Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
Brief introduction of Western blot
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.
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Western blotting is a technique 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 target protein. The target protein is first separated by SDS-PAGE gel electrophoresis. It is then transferred to a membrane and detected using labeled antibodies specific to the target protein. This allows identification and analysis of the target protein in the sample.
Western blotting
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This document discusses different blotting techniques used to detect proteins, DNA, and RNA. It focuses on the Western blot technique used to detect specific proteins. The Western blot process involves tissue preparation, gel electrophoresis to separate proteins, transferring proteins to a membrane, blocking the membrane, primary and secondary antibody detection to reveal targeted proteins, and analysis. Detection can be done through colorimetric, chemiluminescent, or radioactive methods. The Western blot is commonly used as a confirmatory test for HIV and hepatitis infections.
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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.
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Western Blot
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.
Blotting techniques
These slides include all the 3 blotting techniques i,e Southern,northern and western.Their principle,procedure and application.
Western blotting
Western blotting is a widely used technique to detect specific proteins in a tissue or cell extract sample. It involves separating proteins by gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are tissue preparation, gel electrophoresis to separate proteins by size, transferring proteins to a membrane, blocking the membrane to prevent nonspecific binding, primary and secondary antibody incubation for detection, and analysis to identify target proteins. It is a sensitive and specific technique used in research and clinical applications like HIV and hepatitis diagnosis.
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Western blotting is a technique used to detect specific proteins in biological samples. It involves separating proteins by electrophoresis, transferring them to a membrane, and using antibodies to identify the target protein. The presentation discusses the basic western blotting procedure and applications like detecting the molecular weight of proteins, analyzing IgG fractions purified from plasma, confirming HIV tests, and diagnosing hepatitis B virus. It also covers troubleshooting common problems, analyzing protein-protein interactions, and prospects for improving and expanding the technique.
M2-Cinical Immunology
Following is my journal documentation during Master's in Biotechnology completed in 2015. I do understand many changes would've occurred in the curriculum since then, but the basics seldom change. Kindly absorb as per your need.
GEL ELECTROPHORESIS WITH BLOT TECHNIQUES.pptx
This document provides an overview of gel electrophoresis and blotting techniques. It describes how gel electrophoresis uses a gel matrix to separate macromolecules like DNA, RNA, and proteins based on size and charge when an electric current is applied. The document then discusses different types of gel electrophoresis including agarose gel electrophoresis, polyacrylamide gel electrophoresis, and starch gel electrophoresis. It also summarizes common blotting techniques like Southern blotting for detecting DNA, Northern blotting for detecting RNA, and Western blotting for detecting proteins. The document provides details on the basic principles and procedures for each technique.
Western Blotting.pdf
Western blotting is a technique used to detect specific proteins in a tissue sample. It involves three main steps - separating proteins by size through gel electrophoresis, transferring them to a membrane, and detecting the target protein using primary and secondary antibodies. The target protein band can then be visualized, allowing identification and quantification of proteins in a complex mixture. Western blotting is commonly used to identify proteins, estimate their size and amount, and diagnose conditions by detecting antibodies against specific antigens.
Immunoblotting-techniques-Class. Biotechnologypptx
Immunoblotting techniques like ELISA, Western blotting, and Southern blotting utilize the binding specificity between antigens and antibodies. Western blotting involves separating protein mixtures by gel electrophoresis, transferring the proteins to a membrane, and detecting specific proteins using labeled antibodies. It is used to detect the presence of target proteins in complex samples. The key steps are tissue lysis and preparation, gel electrophoresis, protein transfer, membrane blocking, primary/secondary antibody probing, and colorimetric detection. This allows visualization and quantification of proteins separated by size on the membrane.
IMMUNOBLOTTING.pptx
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.
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Time management
Managing time effectively provides several benefits:
It improves efficiency and productivity while lowering stress. Good time management allows people to complete more in less time and achieve goals faster. It also gives people extra free time and leads to more career opportunities since employers value those skills. In contrast, poor time management results in wasted time, loss of control, lower quality work, and a poor reputation.
Radio active labeling
This document discusses radioactive labeling and measurement of radioactivity. It begins by defining atoms and radioactive atoms. It then discusses ionizing radiation and radioactive isotopes. Common radioactive isotopes like carbon-14, tritium, sulfur-35, and phosphorus-32 are described along with their uses. Methods of detecting and measuring radioactivity are summarized, including Geiger Muller counters, scintillation counting, and liquid scintillation counting. Uses of radioactivity in areas like tracing metabolic processes and diagnosing diseases are also covered at a high level.
Micriobiology & Microbes
This document provides an overview of microbiology and microbes. It defines microbes as including bacteria, fungi, algae, viruses, protozoa, and actinomycetes. It describes key early discoveries in microbiology including the invention of the microscope, disproving spontaneous generation, and demonstrating that microorganisms cause disease. It also summarizes techniques that advanced the study of microbiology such as microscopy, sterilization, and using pure cultures.
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.
SDS PAGE
SDS-PAGE is a technique used to separate proteins by size. Proteins are first denatured and treated with SDS to give each polypeptide chain a negative charge proportional to its molecular weight. The polypeptide-SDS complexes are then electrophoresed through a polyacrylamide gel, allowing separation based on size. Smaller complexes migrate farther and faster through the gel than larger ones. Bands are then visualized after staining to reveal the protein sizes.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC is a technique that uses pumps to pass a pressurized liquid solvent containing a sample mixture through a column filled with adsorbent material. This causes the different components in the sample to interact slightly differently with the adsorbent, leading to separation as they flow out of the column. HPLC provides high-speed and high-performance separation compared to traditional chromatography through the use of high pressure pumps and smaller particle size adsorbents. Key components of an HPLC system include the pump, injector, column, detector, recorder, and optional devices like a degasser and column heater.
Dialysis
Dialysis is a process that uses a semi-permeable membrane to selectively remove small molecules from a sample based on size. The membrane allows small molecules like salts to diffuse out of a dialysis bag containing the sample solution and into the surrounding buffer solution. This process is used to desalt and purify protein solutions by removing salts and other small contaminants from the sample inside the dialysis bag until equilibrium is reached. The rate of dialysis is affected by factors like temperature, concentration gradient, membrane thickness, and solvent used.
Lowry's method
This document outlines Lowry's method for determining the total protein content in a wheat flour sample. The method involves forming a complex between amino acids and copper sulfate in an alkaline solution, which is then reduced by phosphomolybdic and phosphotungstic acids to produce a blue color. The intensity of this color can be measured spectrophotometrically and is proportional to the protein content. The document details the reagents, sample preparation by precipitation and washing, using a BSA standard curve to quantify the unknown protein content in the wheat flour sample.
Bradford's method
This document describes Bradford's method for determining the concentration of protein in a serum sample. The method uses Coomassie Brilliant Blue G-250 dye, which binds to protein and shifts its absorption maximum from 465nm to 595nm, allowing for colorimetric quantification. Various volumes of a bovine serum albumin protein standard solution are mixed with dye to generate a standard curve. Unknown serum sample volumes are also mixed with dye and measured at 595nm to determine protein concentration based on the standard curve. The method involves preparing stock and working solutions of dye and protein standard.
Ammonium sulphate precipitation
Ammonium sulphate precipitation is a technique that exploits differences in protein solubility. As ammonium sulphate concentration increases, water molecules hydrate salt ions instead of protein surfaces. This exposes hydrophobic protein regions, causing aggregation and precipitation. Proteins precipitate over a narrow range of salt concentrations, allowing selective enrichment. A sample can be fractionated by successive precipitation steps at increasing ammonium sulphate percentages, isolating proteins based on their solubility thresholds.
Collection of blood, serum & plasma
Blood is a specialized body fluid comprising plasma, red blood cells, white blood cells, and platelets. It makes up about 7-8% of total body weight. Blood collection requires sterile syringes and collection tubes. Serum is blood plasma without fibrinogens and clotting factors, used in diagnostic tests. To obtain serum, blood is collected and centrifuged to separate the clot from supernatant serum. Plasma is the yellow liquid component of blood, comprising about 55% water and dissolved proteins, glucose, and ions. To obtain plasma, blood is centrifuged to separate cells from the plasma supernatant.
Sub cellular fractionation
This document discusses techniques for sub-cellular fractionation, specifically differential velocity centrifugation and density gradient centrifugation. Differential velocity centrifugation separates cellular components based on size, shape, and density by centrifuging at progressively higher speeds. Density gradient centrifugation separates components based on density by layering a cell extract on top of a buffer solution with a gradient of increasing density, allowing components to separate out as they migrate to the position matching their own density. The document provides details on procedures and materials needed to perform sub-cellular fractionation using these techniques.
Cells of the immune system
1. Monocytes and macrophages are part of the immune system's mononuclear cells. Monocytes circulate in the blood before migrating into tissues and differentiating into macrophages.
2. Macrophages are larger than monocytes and have more organelles that allow them to carry out phagocytosis and antigen presentation more effectively. They are found in various tissues throughout the body where they play important roles in innate immunity.
3. Macrophages recognize and phagocytose pathogens via pattern recognition receptors like opsonic receptors, toll-like receptors, and scavenger receptors. Opsonic receptors bind pathogens that have been opsonized with antibodies or complement proteins to enhance phagocytosis.
Cells of the immune system
1. The document describes different types of immune cells including lymphocytes, mononuclear cells, and granulocytic cells.
2. It focuses on granulocytic cells like neutrophils, eosinophils, and basophils. Neutrophils are the most abundant white blood cells and fight pathogens using phagocytosis and releasing contents from granules.
3. Eosinophils and basophils play roles in allergic responses by releasing mediators like histamine from granules when activated. Mast cells are also involved in allergic reactions and contain mediators in granules.
Dinitro salicylic acid (DNSA) method
The document describes a Dinitrosalicylicacid (DNSA) method for testing sugar concentration using a colorimetric assay. Various concentrations of a working sugar standard from 400-2000 μg are prepared and reacted with DNSA reagent and boiled, then the optical density of each sample is measured at 540 nm to generate a standard curve relating sugar concentration to absorbance.
Cells of the immune system
1. Dendritic cells are antigen-presenting cells that can be divided into several types based on location, including Langerhans cells in the skin, interstitial dendritic cells in tissues, and circulating dendritic cells in blood and lymph nodes.
2. Dendritic cells are the most efficient antigen-presenting cells as they constitutively express high levels of MHC class II and co-stimulatory molecules. They take up, process, and present antigens to activate T helper cells.
3. Langerhans cells in the skin take up antigens and migrate to lymph nodes to activate T cells. Interstitial dendritic cells are found in tissues and play a role in immune responses
Cells of the immune system
1) Macrophages phagocytose pathogens through chemotaxis to the site of infection and adherence of pathogens. Opsonins like antibodies and complement proteins enhance phagocytosis.
2) Pattern recognition receptors on macrophages recognize pathogen associated molecular patterns to initiate phagocytosis.
3) Upon phagocytosis, pathogens are contained within phagosomes that fuse with lysosomes, exposing pathogens to digestive enzymes and reactive oxygen species that kill the pathogens.
Cells of the immune system
1. The document describes the major cells of the immune system including lymphocytes like T cells, B cells, and NK cells as well as other immune cells like mast cells, monocytes, macrophages, and dendritic cells.
2. It provides details on the roles and functions of key lymphocyte cells - B cells act as antigen presenting cells, T cells mature in the thymus and have T cell receptors (TCR) while B cells have antibodies, NK cells eliminate tumor and infected cells.
3. The activation and roles of T cell subsets are summarized - TH cells activate through antigen and MHC presentation and secrete cytokines, TC cells are MHC class I restricted and differentiate into cytotoxic T cells, regulatory T cells
Cells of the immune system
This document discusses the cells and organs of the immune system. It describes the primary and secondary lymphoid organs that support lymphocyte development and activation. The primary lymphoid organs are the thymus and bone marrow, which provide an environment for lymphocyte maturation. The secondary lymphoid organs, such as lymph nodes and spleen, are sites where mature lymphocytes encounter antigens. The document also outlines hematopoiesis, the process by which stem cells in the bone marrow differentiate into various immune cells through the influence of cytokines and growth factors in the bone marrow microenvironment.
Sulphadiazine
Sulfadiazine is a synthetic sulphonamide derivative that acts as a competitive inhibitor of p-aminobenzoic acid in the folic acid metabolism cycle of bacteria, inhibiting their multiplication. It is readily absorbed orally but parenteral administration is difficult due to its alkaline nature. Sulfadiazine is widely distributed throughout tissues, achieving high levels in fluids like the pleural, peritoneal, synovial and ocular fluids. It is used in combination with pyrimethamine to treat toxoplasmosis in AIDS patients and newborns with congenital infections.
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BLOTTING
- 2. The term “blotting” refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Southern blot is used for transferring DNA, Northern blot for RNA and Western blot for Protein. Western blotting (also called immunoblotting, because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. Western blotting can produce qualitative and semi-quantitative data about the protein of interest. It is an important technique used in cell and molecular biology. It enables the researchers to identify the specific protein from mixture of proteins extracted from cells as well as evaluation of their size and amount. The SDS PAGE technique is prerequisite for western blotting.
- 3. Principle Western blotting (protein blotting or immunoblotting) is a rapid and sensitive assay for detection and characterization of proteins. It is based on the principle of immunochromatography where proteins are separated into polyacrylamide gel according to their molecular weight. The protein thus separated are then transferred or electro transferred onto nitrocellulose membrane and are detected using specific primary antibody and secondary enzyme labeled antibody and substrate. Western blotting technique is used for identification of particular protein from the mixture of protein. In this method labelled antibody against particular protein is used identify the desired protein, so it is a specific test. Western blotting is also known as immunoblotting because it uses antibodies to detect the protein.
- 5. Procedure Extraction of protein Gel electrophoresis: SDS PAGE Blotting: electrical or capillary blotting Blocking: BSA Treatment with primary antibody Treatment with secondary antibody( enzyme labelled anti Ab) Treatment with specific substrate; if enzyme is alkaline phosphatase, substrate is p-nitro phenyl phosphate which give color.
- 7. Step I: Extraction of Protein Cell lysate is most common sample for western blotting. Protein is extracted from cell by mechanical or chemical lysis of cell. This step is also known as tissue preparation. To prevent denaturing of protein protease inhibitor is used. The concentration of protein is determined by spectroscopy. When sufficient amount of protein sample is obtained, it is diluted in loading buffer containing glycerol which helps to sink the sample in well. Tracking dye (bromothymol blue) is also added in sample to monitor the movement of proteins.
- 8. Step II: Gel electrophoresis The sample is loaded in well of SDS-PAGE Sodium dodecyl sulfate- poly- acrylamide gel electrophoresis. The proteins are separated on the basis of electric charge, isoelectric point, molecular weight, or combination of these all. The small size protein moves faster than large size protein. Protein are negatively charged, so they move toward positive (anode) pole as electric current is applied.
- 9. Step III: Blotting The nitrocellulose membrane is placed on the gel. The separated protein from gel get transferred to nitrocellulose paper by capillary action. This type of blotting is time consuming and may take 1-2 days For fast and more efficient transfer of desired protein from the gel to nitrocellulose paper electro-blotting can be used. In electro-blotting nitrocellulose membrane is sandwich between gel and cassette of filter paper and then electric current is passed through the gel causing transfer of protein to the membrane.
- 10. Step IV: Blocking Blocking is very important step in western blotting. Antibodies are also protein so they are likely to bind the nitrocellulose paper. So before adding the primary antibody the membrane is non- specifically saturated or masked by using casein or Bovine serum albumin (BSA). Step V: Treatment with Primary Antibody The primary antibody (1° Ab) is specific to desired protein so it form Ag- Ab complex
- 11. Step VI: Treatment with secondary antibody The secondary antibody is enzyme labelled. For eg. alkaline phosphatase or Horseradish peroxidase (HRP) is labelled with secondary antibody. Secondary antibody (2° Ab) is antibody against primary antibody (anti-antibody) so it can bind with Ag-Ab complex. Step VII: Treatment with suitable substrate To visualize the enzyme action, the reaction mixture is incubated with specific substrate. The enzyme convert the substrate to give visible colored product, so band of color can be visualized in the membrane. Western blotting is also a quantitative test to determine the amount of protein in sample.