Protein engineering is a branch of biotechnology that modifies protein structure using genetic engineering techniques. The goals of protein engineering include developing proteins with useful functions for medicine, industry, and agriculture by making targeted changes to amino acids based on a protein's 3D structure. Common methods for protein engineering include site-directed mutagenesis and evolutionary methods involving random mutagenesis and selection. Proteome analysis studies all the proteins expressed by a genome at a given time using techniques like protein isolation, separation by SDS-PAGE or IEF, and identification through mass spectrometry.
The document discusses protein engineering and techniques used for it. Protein engineering involves altering cloned DNA to modify protein properties. It merges molecular biology, protein chemistry, and other disciplines. Techniques include genetic modifications like site-directed mutagenesis and chemical modifications. Site-directed mutagenesis allows specific changes to the DNA base using methods like oligonucleotide primers and PCR. This allows investigation of protein function and commercial applications like creating detergent-stable enzymes. Protein engineering has applications in increasing stability, activity and investigating protein properties.
This document discusses protein engineering techniques for modifying proteins, including rational protein design using site-directed mutagenesis and directed evolution using random mutagenesis. Site-directed mutagenesis involves introducing point mutations in a particular known area to modify a specific protein function, while directed evolution generates genetic diversity through random mutagenesis and screens variants to identify successful mutations without requiring structural information. Common random mutagenesis methods discussed are error-prone PCR and DNA shuffling, which can be used to engineer properties like protein folding, stability, binding, and catalysis.
Principal of genetic engineering & its applications inlaraib jameel
Genetic engineering involves directly manipulating an organism's DNA. It can introduce DNA from other species into a host organism, creating genetically modified organisms (GMOs). Early applications included creating bacteria that produced human insulin to treat diabetes. Now genetic engineering is used widely in scientific research and agriculture to improve crop yields and traits. It also has medical applications like developing model organisms to study diseases, producing therapeutic proteins and antibodies, and holding promise for future gene therapies and regenerative medicine using stem cells.
Multiple proteins are required for DNA replication, including DNA polymerases, helicases, primase, topoisomerases, ligase and single-stranded DNA binding proteins. Helicases unwind DNA at replication forks using ATP while primase synthesizes RNA primers and DNA polymerases use the primers to replicate DNA in the 5' to 3' direction. DNA gyrase introduces negative supercoils to relieve positive supercoiling formed during unwinding while ligase seals nicks between Okazaki fragments to complete replication.
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
This document discusses protein engineering and methods to improve protein stability. It begins by defining protein engineering as modifying protein structure using recombinant DNA technology or chemical treatment to achieve desirable functions. The objectives of protein engineering are outlined, such as creating superior enzymes for industrial use. Methods of protein engineering discussed include mutagenesis and gene modification techniques. Strategies to increase protein stability through additions like disulfide bonds or changes to amino acids are also presented. The document provides examples of protein engineering applications in medicine, industry and agriculture.
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
Recombinant DNA technology involves manipulating DNA sequences in the laboratory. DNA is isolated, cut with restriction enzymes, and joined with DNA ligase. The recombinant DNA is inserted into a cloning vector and introduced into a host cell. Cells containing the recombinant DNA are selected and amplified. This allows large quantities of identical DNA molecules to be produced for analysis, comparison, and other purposes. Common applications include understanding disease, producing therapeutic proteins, disease prevention through vaccines, diagnosis, and gene therapy.
The document discusses protein engineering and techniques used for it. Protein engineering involves altering cloned DNA to modify protein properties. It merges molecular biology, protein chemistry, and other disciplines. Techniques include genetic modifications like site-directed mutagenesis and chemical modifications. Site-directed mutagenesis allows specific changes to the DNA base using methods like oligonucleotide primers and PCR. This allows investigation of protein function and commercial applications like creating detergent-stable enzymes. Protein engineering has applications in increasing stability, activity and investigating protein properties.
This document discusses protein engineering techniques for modifying proteins, including rational protein design using site-directed mutagenesis and directed evolution using random mutagenesis. Site-directed mutagenesis involves introducing point mutations in a particular known area to modify a specific protein function, while directed evolution generates genetic diversity through random mutagenesis and screens variants to identify successful mutations without requiring structural information. Common random mutagenesis methods discussed are error-prone PCR and DNA shuffling, which can be used to engineer properties like protein folding, stability, binding, and catalysis.
Principal of genetic engineering & its applications inlaraib jameel
Genetic engineering involves directly manipulating an organism's DNA. It can introduce DNA from other species into a host organism, creating genetically modified organisms (GMOs). Early applications included creating bacteria that produced human insulin to treat diabetes. Now genetic engineering is used widely in scientific research and agriculture to improve crop yields and traits. It also has medical applications like developing model organisms to study diseases, producing therapeutic proteins and antibodies, and holding promise for future gene therapies and regenerative medicine using stem cells.
Multiple proteins are required for DNA replication, including DNA polymerases, helicases, primase, topoisomerases, ligase and single-stranded DNA binding proteins. Helicases unwind DNA at replication forks using ATP while primase synthesizes RNA primers and DNA polymerases use the primers to replicate DNA in the 5' to 3' direction. DNA gyrase introduces negative supercoils to relieve positive supercoiling formed during unwinding while ligase seals nicks between Okazaki fragments to complete replication.
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
This document discusses protein engineering and methods to improve protein stability. It begins by defining protein engineering as modifying protein structure using recombinant DNA technology or chemical treatment to achieve desirable functions. The objectives of protein engineering are outlined, such as creating superior enzymes for industrial use. Methods of protein engineering discussed include mutagenesis and gene modification techniques. Strategies to increase protein stability through additions like disulfide bonds or changes to amino acids are also presented. The document provides examples of protein engineering applications in medicine, industry and agriculture.
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
Recombinant DNA technology involves manipulating DNA sequences in the laboratory. DNA is isolated, cut with restriction enzymes, and joined with DNA ligase. The recombinant DNA is inserted into a cloning vector and introduced into a host cell. Cells containing the recombinant DNA are selected and amplified. This allows large quantities of identical DNA molecules to be produced for analysis, comparison, and other purposes. Common applications include understanding disease, producing therapeutic proteins, disease prevention through vaccines, diagnosis, and gene therapy.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
This presentation gives an brief idea about the applications of genetic engineering which is of at most importance to humans. Provided along with this slide is an example which makes it easier to understand the concept.
Protein engineering involves modifying protein structure using recombinant DNA technology or chemical treatment to improve function for use in medicine, industry, and agriculture. The objectives of protein engineering are to create superior enzymes for specific chemical production, produce enzymes in large quantities, and produce superior biological compounds. Protein engineering aims to alter properties like kinetic properties, thermostability, stability in nonaqueous solvents, substrate specificity, and cofactor requirements to meet industrial needs. Common methods for protein engineering include mutagenesis, selection, and recombinant DNA technology.
Recombinant DNA (rDNA) technology involves combining DNA molecules from different sources into a single recombinant DNA molecule. This is done using restriction enzymes to cut the DNA at specific sites and DNA ligase to join the fragments. The resulting recombinant DNA can be inserted into a host cell that will replicate it, allowing mass production of useful proteins like insulin, growth hormone, and monoclonal antibodies for therapeutic use. While rDNA technology has generated many medical advances, it also raises safety and ethical concerns that must be carefully considered and addressed.
Genetic engineering involves directly manipulating genes, often by adding a gene from another species to an organism's genome. This is done through recombinant DNA (rDNA) technology, which combines DNA sequences artificially. A key part of the process is using restriction enzymes to cut DNA at specific sites, then inserting the cut DNA fragment into a vector like a plasmid for replication in a host cell. The engineered DNA is then introduced into host cells, and cells containing the new DNA are identified and isolated through markers on the vector.
This document summarizes different types of DNA ligase enzymes including their sources, mechanisms, and applications. It discusses bacteriophage T4 DNA ligase, E.coli DNA ligase, Taq DNA ligase, T4 RNA ligase, and mammalian ligases. The mechanism of DNA ligase involves three steps - adenylation of the enzyme using ATP, transfer of AMP to the DNA donor strand, and formation of a phosphodiester bond between the donor and acceptor strands. The types of ligases vary in their substrate specificities and thermal stabilities, making each useful for different molecular biology applications like cloning and DNA amplification.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
This document discusses various techniques in microbial genetics including transformation, transduction, conjugation, plasmids, and transposons. Transformation involves the uptake of genetic material like DNA by bacterial cells. Transduction occurs when viruses called bacteriophages transfer genetic material between bacteria. Conjugation is the transfer of genetic material like plasmids through direct contact between bacteria. Plasmids are small circular DNA molecules that are distinct from chromosomal DNA and often provide genetic advantages to bacteria. Transposons are genetic elements that can move to different locations in a genome and contribute to the spread of traits like antibiotic resistance.
Genetic organization of eukaryotes and prokaryotes Theabhi.in
The document discusses the genetic organization of prokaryotes and eukaryotes. Prokaryotes typically have a single circular chromosome, while eukaryotes have multiple linear chromosomes contained within a nucleus. The prokaryotic genome is much smaller than the eukaryotic genome. Key differences include prokaryotes lacking membrane-bound organelles and having genes that are not interrupted by non-coding sequences like introns.
The document discusses drug design, development, and delivery. It covers rational drug design using molecular properties and receptor modeling. Computer-assisted drug design uses molecular docking and QSAR methods. Neural networks are also used in drug design. Drug discovery involves identifying candidates and screening for efficacy. Drug development evaluates ADME, toxicity, and safety through preclinical and clinical studies. Drug delivery methods aim to effectively administer pharmaceutical compounds and improve drug release profiles.
Cosmid Vector and Yeast artificial chromosome Vector and Plant Vectors ( Ti ...Amany Elsayed
1. Cosmid vectors are cloning vectors derived from bacteriophages that can contain up to 44 kilobase pairs of foreign DNA. They are commonly used to clone large fragments of genomic DNA in E. coli.
2. Yeast artificial chromosomes (YACs) are engineered chromosomes used to clone DNA in yeast cells. They contain telomeres, a centromere, autonomous replicating sequences, and selectable markers to replicate and maintain cloned DNA.
3. Plant vectors use the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens as the primary vector. The Ti plasmid transfers T-DNA containing the gene of interest into the plant genome, allowing genetic modification of
This document discusses protein engineering, which uses recombinant DNA technology to modify protein structure and function. It describes several methods of protein engineering, including site-directed mutagenesis, error-prone PCR, and DNA shuffling. The objectives of protein engineering are to improve protein stability, modify cofactor requirements, increase enzyme activity, and modify enzyme specificity. As an example, the document discusses how site-directed mutagenesis was used to increase the stability of streptokinase by replacing lysine residues susceptible to cleavage by plasmin.
This document contains lecture notes on major histocompatibility complex (MHC) and related topics from a biotechnology course. It discusses antigen-presenting cells, the structure and function of MHC class I and II molecules, similarities and differences between the two classes, MHC-associated genes, and important immune signaling molecules like cytokines, interleukins, interferons, and chemokines. Diagrams are included to illustrate MHC pathway and types of interferons. The notes provide an overview of key concepts in MHC and immunology for students in the biotechnology course.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
Genome organization in prokaryotes(molecular biology)IndrajaDoradla
1. In prokaryotes, the genome is located in an irregularly shaped region within the cell called the nucleoid, which is not surrounded by a membrane like the eukaryotic nucleus.
2. The prokaryotic genome is generally a circular piece of DNA that can exist in multiple copies and ranges in length but is at least a few million base pairs. It is packaged into the nucleoid through supercoiling facilitated by nucleoid-associated proteins.
3. DNA supercoiling allows for very long strands of DNA to be tightly packaged into a prokaryotic cell. This involves the introduction of plectonemic supercoils that twist the DNA into loops and wind it around nucle
PCR (polymerase chain reaction) is an in vitro technique used to amplify a specific region of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample to separate the DNA strands and allow primers to anneal, followed by extension of the primers by a thermostable DNA polymerase. Kary Mullis developed PCR in 1985 and was awarded the Nobel Prize for Chemistry in 1993. Requirements for PCR include a DNA template, primers, Taq polymerase enzyme, dNTPs, buffer solution and magnesium ions. There are several applications and variations of PCR including quantitative real-time PCR, reverse transcription PCR, and inverse PCR.
This document discusses gene identification and discovery. It begins by describing gene identification in prokaryotes, unicellular eukaryotes, and multicellular eukaryotes. It then discusses the components of protein-coding genes and different approaches to gene prediction for prokaryotes and eukaryotes. The document also covers gene structure in prokaryotes and eukaryotes, as well as software tools and methods for gene prediction. Finally, it discusses several approaches to classifying genes by function.
This document provides information on genomics, proteomics, and metabolomics. It discusses that genomics is the study of genomes through sequencing and analysis. It involves various types of genomics like structural, functional, and comparative genomics. Proteomics is the large-scale study of the structure and function of proteins in organisms. Key proteomics methods include antibody detection and mass spectrometry. Metabolomics is the study of small molecule metabolites within cells and biofluids, which make up the metabolome. These "omics" fields provide insights into cellular processes and are applied in areas like disease diagnosis and drug development.
Proteomics and its applications in phytopathologyAbhijeet Kashyap
Dear friends, I Abhijeet kashyap presenting the basics of proteomics to you all . Proteomics is the large-scale study of proteins, particularly their structures and functions.Proteomics helps in understanding the structure and function of different proteins as well as protein-protein interactions of an organism.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
This presentation gives an brief idea about the applications of genetic engineering which is of at most importance to humans. Provided along with this slide is an example which makes it easier to understand the concept.
Protein engineering involves modifying protein structure using recombinant DNA technology or chemical treatment to improve function for use in medicine, industry, and agriculture. The objectives of protein engineering are to create superior enzymes for specific chemical production, produce enzymes in large quantities, and produce superior biological compounds. Protein engineering aims to alter properties like kinetic properties, thermostability, stability in nonaqueous solvents, substrate specificity, and cofactor requirements to meet industrial needs. Common methods for protein engineering include mutagenesis, selection, and recombinant DNA technology.
Recombinant DNA (rDNA) technology involves combining DNA molecules from different sources into a single recombinant DNA molecule. This is done using restriction enzymes to cut the DNA at specific sites and DNA ligase to join the fragments. The resulting recombinant DNA can be inserted into a host cell that will replicate it, allowing mass production of useful proteins like insulin, growth hormone, and monoclonal antibodies for therapeutic use. While rDNA technology has generated many medical advances, it also raises safety and ethical concerns that must be carefully considered and addressed.
Genetic engineering involves directly manipulating genes, often by adding a gene from another species to an organism's genome. This is done through recombinant DNA (rDNA) technology, which combines DNA sequences artificially. A key part of the process is using restriction enzymes to cut DNA at specific sites, then inserting the cut DNA fragment into a vector like a plasmid for replication in a host cell. The engineered DNA is then introduced into host cells, and cells containing the new DNA are identified and isolated through markers on the vector.
This document summarizes different types of DNA ligase enzymes including their sources, mechanisms, and applications. It discusses bacteriophage T4 DNA ligase, E.coli DNA ligase, Taq DNA ligase, T4 RNA ligase, and mammalian ligases. The mechanism of DNA ligase involves three steps - adenylation of the enzyme using ATP, transfer of AMP to the DNA donor strand, and formation of a phosphodiester bond between the donor and acceptor strands. The types of ligases vary in their substrate specificities and thermal stabilities, making each useful for different molecular biology applications like cloning and DNA amplification.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
This document discusses various techniques in microbial genetics including transformation, transduction, conjugation, plasmids, and transposons. Transformation involves the uptake of genetic material like DNA by bacterial cells. Transduction occurs when viruses called bacteriophages transfer genetic material between bacteria. Conjugation is the transfer of genetic material like plasmids through direct contact between bacteria. Plasmids are small circular DNA molecules that are distinct from chromosomal DNA and often provide genetic advantages to bacteria. Transposons are genetic elements that can move to different locations in a genome and contribute to the spread of traits like antibiotic resistance.
Genetic organization of eukaryotes and prokaryotes Theabhi.in
The document discusses the genetic organization of prokaryotes and eukaryotes. Prokaryotes typically have a single circular chromosome, while eukaryotes have multiple linear chromosomes contained within a nucleus. The prokaryotic genome is much smaller than the eukaryotic genome. Key differences include prokaryotes lacking membrane-bound organelles and having genes that are not interrupted by non-coding sequences like introns.
The document discusses drug design, development, and delivery. It covers rational drug design using molecular properties and receptor modeling. Computer-assisted drug design uses molecular docking and QSAR methods. Neural networks are also used in drug design. Drug discovery involves identifying candidates and screening for efficacy. Drug development evaluates ADME, toxicity, and safety through preclinical and clinical studies. Drug delivery methods aim to effectively administer pharmaceutical compounds and improve drug release profiles.
Cosmid Vector and Yeast artificial chromosome Vector and Plant Vectors ( Ti ...Amany Elsayed
1. Cosmid vectors are cloning vectors derived from bacteriophages that can contain up to 44 kilobase pairs of foreign DNA. They are commonly used to clone large fragments of genomic DNA in E. coli.
2. Yeast artificial chromosomes (YACs) are engineered chromosomes used to clone DNA in yeast cells. They contain telomeres, a centromere, autonomous replicating sequences, and selectable markers to replicate and maintain cloned DNA.
3. Plant vectors use the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens as the primary vector. The Ti plasmid transfers T-DNA containing the gene of interest into the plant genome, allowing genetic modification of
This document discusses protein engineering, which uses recombinant DNA technology to modify protein structure and function. It describes several methods of protein engineering, including site-directed mutagenesis, error-prone PCR, and DNA shuffling. The objectives of protein engineering are to improve protein stability, modify cofactor requirements, increase enzyme activity, and modify enzyme specificity. As an example, the document discusses how site-directed mutagenesis was used to increase the stability of streptokinase by replacing lysine residues susceptible to cleavage by plasmin.
This document contains lecture notes on major histocompatibility complex (MHC) and related topics from a biotechnology course. It discusses antigen-presenting cells, the structure and function of MHC class I and II molecules, similarities and differences between the two classes, MHC-associated genes, and important immune signaling molecules like cytokines, interleukins, interferons, and chemokines. Diagrams are included to illustrate MHC pathway and types of interferons. The notes provide an overview of key concepts in MHC and immunology for students in the biotechnology course.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
Genome organization in prokaryotes(molecular biology)IndrajaDoradla
1. In prokaryotes, the genome is located in an irregularly shaped region within the cell called the nucleoid, which is not surrounded by a membrane like the eukaryotic nucleus.
2. The prokaryotic genome is generally a circular piece of DNA that can exist in multiple copies and ranges in length but is at least a few million base pairs. It is packaged into the nucleoid through supercoiling facilitated by nucleoid-associated proteins.
3. DNA supercoiling allows for very long strands of DNA to be tightly packaged into a prokaryotic cell. This involves the introduction of plectonemic supercoils that twist the DNA into loops and wind it around nucle
PCR (polymerase chain reaction) is an in vitro technique used to amplify a specific region of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample to separate the DNA strands and allow primers to anneal, followed by extension of the primers by a thermostable DNA polymerase. Kary Mullis developed PCR in 1985 and was awarded the Nobel Prize for Chemistry in 1993. Requirements for PCR include a DNA template, primers, Taq polymerase enzyme, dNTPs, buffer solution and magnesium ions. There are several applications and variations of PCR including quantitative real-time PCR, reverse transcription PCR, and inverse PCR.
This document discusses gene identification and discovery. It begins by describing gene identification in prokaryotes, unicellular eukaryotes, and multicellular eukaryotes. It then discusses the components of protein-coding genes and different approaches to gene prediction for prokaryotes and eukaryotes. The document also covers gene structure in prokaryotes and eukaryotes, as well as software tools and methods for gene prediction. Finally, it discusses several approaches to classifying genes by function.
This document provides information on genomics, proteomics, and metabolomics. It discusses that genomics is the study of genomes through sequencing and analysis. It involves various types of genomics like structural, functional, and comparative genomics. Proteomics is the large-scale study of the structure and function of proteins in organisms. Key proteomics methods include antibody detection and mass spectrometry. Metabolomics is the study of small molecule metabolites within cells and biofluids, which make up the metabolome. These "omics" fields provide insights into cellular processes and are applied in areas like disease diagnosis and drug development.
Proteomics and its applications in phytopathologyAbhijeet Kashyap
Dear friends, I Abhijeet kashyap presenting the basics of proteomics to you all . Proteomics is the large-scale study of proteins, particularly their structures and functions.Proteomics helps in understanding the structure and function of different proteins as well as protein-protein interactions of an organism.
role of proteomics in target discovery and validation
1 target of drug action
2 proteomics
3 facts about proteins
4 post translational modification
5 additional modification
6 methods of studying proteins
7 hybrid technologies
Introduction to proteomics, techniques to study proteomics such as protein electrophoresis, chromatography and mass spectrometry and protein database analysis, case studies derived from scientific literature including comparisons between healthy and diseased tissues, new approaches to analyse metabolic pathways, comprehensive analysis of protein-protein interactions in different cell types.
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.
This document discusses various applications of protein engineering in different industries and fields. It describes how protein engineering is used in the food industry to modify enzymes like proteases, amylases, and lipases to improve their properties. It also discusses applications in environmental remediation, medicine like cancer treatment, biopolymer production, nanobiotechnology, and redox proteins. The document provides an overview of the wide range of uses of protein engineering across diverse domains.
Meta-genomics is the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”
Biotechnology is the use of living organisms to develop products and technologies. Pharmaceutical biotechnology applies biotechnology principles to develop drugs. The majority of current drugs are biologics such as antibodies, nucleic acids, and vaccines. Biotechnology methods are important in drug research and development, with key applications in oncology, metabolic disorders, and musculoskeletal disorders. Examples of biotech drugs include insulin for diabetes, gene therapy to replace mutated genes, clotting factors for hemophilia, human serum albumin for burns treatment, and engineered enzymes for enzyme deficiencies.
Proteomics is the study of the proteome, which is the entire set of proteins expressed by a genome, cell, tissue or organism. This document discusses several techniques used in proteomics including 2D gel electrophoresis, mass spectrometry, and protein databases. It provides examples of applications such as biomarker identification for disease diagnosis and drug target discovery. Limitations include the complexity of proteomes and no single technique being adequate for complete analysis. Overall, proteomics techniques help further our understanding of protein structure, function and interactions to gain insights into biological processes and diseases.
Microorganisms such as bacteria, actinomycetes, and fungi are ubiquitous on our planet. They are widely distributed in soil, water, the human body and other environments. Microorganisms and their activities are of great importance to biogeochemical cycles and to all biological systems. Creative Proteomics provides a one-stop proteomics service from sample collection, protein separation, to protein quantification and bioinformatics analysis. We offer both relative quantification (including iTRAQ, TMT and SILAC) and absolute quantification (such as SRM/MRM and PRM) approaches to help you discover, detect and quantify proteins in a broad array of samples. https://www.creative-proteomics.com/services/proteomics-service.htm
Microorganisms such as bacteria, actinomycetes, and fungi are ubiquitous on our planet. They are widely distributed in soil, water, the human body and other environments. Microorganisms and their activities are of great importance to biogeochemical cycles and to all biological systems. Creative Proteomics provides a one-stop proteomics service from sample collection, protein separation, to protein quantification and bioinformatics analysis. We offer both relative quantification (including iTRAQ, TMT and SILAC) and absolute quantification (such as SRM/MRM and PRM) approaches to help you discover, detect and quantify proteins in a broad array of samples. https://www.creative-proteomics.com/services/proteomics-service.htm
Structural genomics aims to determine the 3D structures of all proteins encoded by genomes through high-throughput methods. It uses a genome-based approach to solve protein structures rapidly and cost-effectively. Major initiatives like the Protein Structure Initiative have made progress in determining thousands of protein structures. Challenges include expressing membrane and eukaryotic proteins, as well as determining remaining novel folds. Determining protein structures through structural genomics increases understanding of protein function and facilitates drug discovery.
This document discusses protein microarrays, which are a high-throughput screening method. Protein microarrays allow researchers to study protein interactions and activities on a large scale by placing many proteins on a solid surface in defined locations. There are two main types of protein microarrays: analytical microarrays which use antibodies or other capture molecules to probe complex protein solutions, and functional microarrays which contain full-length functional proteins to study protein-protein and other biochemical interactions. Protein microarrays have applications in areas like biomarker identification, studying post-translational modifications, and host-pathogen interactions. However, several challenges remain in developing stable and sensitive protein microarrays.
Designing of drug delivery system for biotechnology products considering stab...Smaranika Rahman
"Where there is life, there is DNA, where there is DNA, there is biotechnology." Biotechnology, as the word suggests, is combination of biology and technology. So the importance of biotechnology and biotechnology products in our life is increasing day by day. That's why we have to produce biotechnology products in a safer manner and also maintain that through it's shelf-life.We have also research on improving methods of improving it's stability. In this topic, I also tried to discuss bioinformatic-driven strategies that are used to predict structural changes that can be applied to wild type proteins in order to produce more stable variants. The most commonly employed techniques PEGylation, stochastic approaches, empirical or systematic rational design strategies.
Drug delivery system for biotech product considering stability aspects and mo...zobaida mostarin nishi
Drug delivery is becoming a whole interdisciplinary and
an independent field of research and is gaining the attention of
pharmaceutical makers, medical doctors, and industry. A
targeted and safe drug delivery could improve the performance
of some classical medicines already on the market and,
moreover, will have implications for the development and
the success of new therapeutic strategies, such as peptide and
protein delivery, glycoprotein administration, gene therapy and
RNA interference.
A comparative study using different measure of filterationpurkaitjayati29
This document presents a study comparing different scoring functions used in filter-based feature selection methods for microarray gene expression data. Chapter 1 introduces gene expression, DNA microarrays, and the goals of classification and feature selection. Chapter 2 provides background on bioinformatics, molecular biology, and the central dogma. Chapter 3 describes DNA microarray technology and gene expression data. Chapter 4 reviews literature on feature selection techniques applied to microarray data, discussing filter, wrapper, embedded, hybrid, and ensemble methods. Chapter 5 proposes using a scoring function-based filter method to select relevant genes, focusing on mutual information, symmetric uncertainty, information gain, and Chi-square scoring functions.
The document discusses protein-protein interactions (PPIs), including an introduction to PPIs, the types of interactions, techniques used to study them like X-ray crystallography, NMR spectroscopy and cryo-electron microscopy, and factors that affect PPIs. It also covers methods to investigate PPIs such as affinity purification coupled with mass spectrometry and yeast two-hybrid screening. Applications of understanding PPIs include developing therapeutic drugs and identifying functions of unknown proteins.
Proteomics: types, protein profiling steps etc.Cherry
Proteome is a set of proteins produced in an organism, system, or biological context or entire set of proteins that is, or can be, expressed by a genome, cell, tissue, or organism at a certain expressed time in a given set of condition. Proteomics is the study of all the proteins produced by a cell.
Proteomics, definatio , general concept, signficanceKAUSHAL SAHU
INTRODUCTION
GENERAL CONCEPT
WHY PROTEIOMIC NECESERY?
WHAT PROTEOMIC CAN ANSWER?
PRTEOMICS- ANALYSIS AND IDENTIFICATION OF PROTEIN
TWO-DIMENSIONAL SDS-PAGE
MASS SPECTROMETERS
SIGNIFICANCE OF STUDY AN ITS IMPORTANCE
APPLICATIONS
CHALLENGES
CONCLUSIONS
REFERENCES
Protein microarrays allow thousands of proteins to be analyzed simultaneously. They consist of a solid surface coated with protein spots that are probed with labeled molecules. There are several types including analytical, functional, and reverse phase arrays. Protein microarrays can be used for applications such as drug discovery, biomarker identification, and clinical diagnostics. They offer advantages over other methods like ELISA in terms of throughput, sensitivity, and cost. Future opportunities include integration with other technologies and development of single-cell and multiplexed protein assays.
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/
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
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.
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
3. • Protein engineering is a branch of biotechnology that falls under a
discipline called synthetic biology.
• Protein Engineering can be defined as the modification of protein
structure with recombinant DNA technology or chemical treatment
to get a desirable function for better use in medicine , industry ,
agriculture , and other fields also.
• Protein engineering is the process of developing useful valuable
protein.
• Protein engineering involves premeditated change of amino acids
and is usually based on the known 3-D structure of a given protein
and its biochemically established catalytic mechanism.
Protein Engineering
4. History
1- In 1951 Fredrick Sanders discovered
how to determine the sequence of amino
acid .
2- Before this it was thought that protein
have no definite structure. This allowed
for translation to be studied as it provided
the framework for DNA coding of protein.
3- In 1983 Kary Mullis developed the PCR.
5. Reason To Engineering A Protein
1- To bring changes in catalytic properties .
2- To bring changes in structural properties.
3- Creation of new system .
6.
7.
8. Objectives / Need of protein engineering
⮚ To create a superior enzyme to catalyze the production of high value specific
chemicals
⮚ To produce enzyme in large quantities.
⮚ To develop useful valuable proteins .
⮚ To get a desirable function for better use in
medicine , industry and agriculture .
9. ⮚ Elimination of allosteric Regulations.
⮚ Improved kinetics properties of
enzymes.
⮚ To produce biological compound
(include synthetic peptides , storage
protein , and synthetic drugs ) superior
to natural one.
10. ⮚ Enhanced substrate and reaction specificity.
⮚ Increased thermostability.
⮚ Alteration in optimum pH.
⮚ Solubility for use in organic solvents.
⮚ Increased / decreased Optimal temperature.
⮚ To speed up the process ( rate of Reaction )
⮚ Increase Protein / Enzymes shelf life.
⮚ To get High quality of product suitability face.
11. Basic assumption for protein engineering
• While doing protein engineering should recognize the
following properties of enzymes,
o Many amino acid substitution , deletions or addition
lead to no changes in enzymes activity so that they
are silent mutator.
o Protein have limited number of basic structures and
only minor changes are superimposed on them
leading to variation.
o Similar patterns of chain folding and domain
structure can arise from different amino acid
sequences with little or no homology.
12. METHODS :
A variety of methods are used in protein engineering such as:
• MUTAGENESIS
• PROTEIN ENGINEERING BY USE OF GENE FAMILIES
• PROTEIN ENGINEERING THROUGH CHEMICAL MODIFICATION.
The most classical method in protein engineering is the so-called “rational design” approach which
involves “site-directed mutagenesis” of proteins.
The use of “evolutionary methods” that involve “random mutagenesis and selection” for the desired
protein properties was introduced as an alternative approach.
“In vitro protein evolution systems” are also important methods in protein engineering.
“Flow cytometry”, a powerful method for single cell analysis, is also used in protein
engineering studies.
13. • Method name Reference(s)
• Rational design (Arnold, 1993)
• Site-directed
mutagenesis (Arnold, 1993), (Antikainen &
Martin, 2005)
• Evolutionary methods/
directed evolution (Arnold, 1993)
• Random mutagenesis (Antikainen & Martin, 2005), (Wong
et al., 2006),
• DNA shuffling (Antikainen & Martin, 2005),
(Jackson et al., 2006)
• Molecular dynamics (Anthonsen et al., 1994)
• Homology modeling (Anthonsen et al., 1994)
14.
15.
16. The most classical method in protein engineering is the so-called
“rational design” approach which involves “site-directed
mutagenesis” of proteins.
17.
18.
19. the use of “evolutionary methods” that involve “random
mutagenesis and selection” for the desired protein properties was
introduced as an alternative approach
20.
21. PROTEIN ENGINEERING BY USE OF GENE
FAMILIES
• This technique involves isolation of gene from
each species and create hybrid for eq-
substillisin ( an enzyme used detergent
industry), genes from 26 species were mixed so
we get 4 types of substilisin enzyme with
improved qualities in different aspect
22. PROTEIN ENGINEERING THROUGH CHEMICAL
MOdIFICATIONS.
• In this method different chemicals used as
“glutaraldehyde”.
• It acts as protein cross linker.
• It stabilizes the protein in solutions.
• For example, insulin, lactate de hydrogenase
23. APPLICATIONS:
A variety of protein engineering applications have been reported in the literature.
These applications range from biocatalysis for food and industry to
environmental, medical and nanobiotechnology applications.
Food and detergent industry applications :
Early reports on the importance of protein engineering methods to design new
enzymes for enzyme biotechnological industries date back to 1993 (Wiseman,
1993).
Particularly, the enzymes used in food industry were emphasized as an
important group of enzymes, the industrially important properties of which could
be further improved by protein engineering.
Environmental applications :
Environmental applications of enzyme and protein engineering are also another
important field.
Early reports on enzyme and cell applications in industry and in environmental
monitoring, such as environmental biosensors, date back to 1993.
24. Medical applications:
Medical applications of protein engineering are also diverse.
The use of protein engineering for cancer treatment studies is a
major area of interest.
Pretargeted radioimmunotherapy has been discussed as a
potential cancer treatment.
By pretargeting, radiation toxicity is minimized by separating the
rapidly cleared radionuclide and the long-circulating antibody.
Advances in protein engineering and recombinant DNA
technology were expected to increase the use of pretargeted
radioimmunotherapy.
26. PROTEOME:
►proteome word coined from 2 words protein & genome by marc Wilkins in 1994.
►proteome refers to the total set of proteins expressed in a given cell at a given time.
►it is defined as the full complement of proteins produced by a particular genome.
►proteome is larger than the genome, especially in eukaryotes, in the sense that there are
more proteins genes.
►the proteome of a cell depends on the cell type,its developmental stage,
environmental/stimuli, nutritional and metabolic status etc.
TYPE OF PROTEOME:
1. CELLULAR PROTEOME
2. COMPLETE PROTEOME
INTRODUCTION
29. PROTEOMIC TOOLS TO STUDY
PROTEINS:
☻PROTEIN ISOLATION: 1-Mechanical method
2-Chemical method
☻PROTEIN SEPARATION: Two methods:
1-SDS-PAGE
2-IEF
☻PROTEIN IDENTIFICATION: Use of Mass Spectrometry
30.
31.
32.
33.
34.
35.
36. WHY PROTEOMICS
• Proteomics is the protein equivalent of genomics and has captured the
imagination of biomolecular scientists worldwide.
• Aimed at determining the identity and quantity of expressed proteins in
cells, their three-dimensional structure.
• For example, it is extensively applied to the study of proteins involved
in carcinogenesis, as well as to discover biomarkers for clinical use,
for screening, diagnosis, staging, prognosis, monitoring response to
treatment, and detection of recurrent diseases.
• The question of how one protein regulates the activity of another by
binding to it requires an integration of structural, functional, and
dynamic information.
37. From Genomics to Proteomics
• It involves the identification of proteins in the body and
the determination of their roles in physiological and
pathophysiological functions.
• Proteins that are directly involved in both normal and
disease-associated biochemical processes
• Genomics does not predict PTM that most proteins
undergo
Human Genome
Project
Protein compliment of the human
organism.
38. PROTEOMICS APPLICATIONS
• Most common application of proteomics is protein
identification.
• To accomplish this, we use affinity columns and other
strategies to select for protein targets.
• Characterization of Protein Complexes:
• Proteomics Approach to Protein Phosphorylation
• Proteome Mining
39. Challenges
• Proteins are more difficult to work with than DNA and
RNA; have secondary and tertiary structure.
• Proteins cannot be amplified like DNA.
• Each proteomics technology can only analyze proteins
within 3–4 orders of magnitude
• Thulasiraman et al. developed the new deep proteome
approach via ligand library beads
40. Perspectives
• Differential proteomics is a scientific discipline that detects
the proteins associated with a disease.
• Proteomics research permits the discovery of new protein
markers for diagnostic purposes.
• Biomarkers may also be used to help devising an optimal
therapeutic treatment plan for different patient subsets and
to monitor the effect of treatment.
• Proteomics has much promise in novel drug discovery.
• Proteomics makes a key contribution to the development of
functional genomics.
42. Mass Spectrometry
• Mass spectrometry (MS) is an analytical technique that produces spectra
(singular spectrum) of the masses of the atoms or molecules comprising a
sample of material
• Mass spectrometry is an extremely valuable analytical technique in which the
molecules in test sample are converted into gaseous ions that are
subsequently separated in a mass spectrometer according to their mass-to-
charge ratio (m/z) and detected
43. Components Of A Mass Spectrometer
❖ Sample input system
❖ Ionization source
❖ Mass analyzer
❖ Detector
❖ Computer based data acquisition and processing system
44. Mass Spectrum
❖ The mass spectrum is a plot of a relative abundance of the ions at
each m/z ratio.
❖ In most cases, the nascent molecular ions of the analyte produce
fragment ions by cleavage of the bond and the resulting
fragmentation pattern constitute the mass spectrum
45. Principle And Instrumentation
1. IONIZATION
❖ Ionization is a process of charging a molecule.
❖ The sample molecule must be charged in order to measure them using a
mass spectrometer
❖ The atom is ionized by knocking one or more electrons off to give a positive
ion.
❖ The particle in the sample (Atom or molecules) bombarded with stream of
electron to knock one or more electron out of the sample particles to make
positive ions.
46. Principle And Instrumentation
2. ACCELERATION
❖ The ions are accelerated so that they all have the same kinetic energy
❖ The positive ions are repelled away from the positive ionization
chamber and pass through 3 slits with voltage in decreasing order.
❖ All the ions are accelerated into freely focused beam.
47. Principle And Instrumentation
3. DEFLECTION
❖ The ions in the deflected by a
magnetic field according to their
masses.
❖ Different ions are deflected by
the magnetic field by different
amounts.
Amount of deflection depends upon
1. The mass of the ion
2. The charge of the ion
48. Principle And Instrumentation
4. Detection
❖ The beam of ions passing through
the machine is detected electrically
❖ Ions leaves space in metal by
neutralizing it & the electron in the
wire shuffle along to fill it.
❖ The flow of electrons in the wire is
detected as an electric current
which can be amplified & recorded A continuous dynode particle multiplier detector.
50. Applications
• Mass spectrometry is also used to determine the isotopic
composition of elements within a sample. Difference in mass
among isotopes of an element are very small, and less abundant
isotopes of an element are typically very rare,
• Mass spectrometry is an important method for the
characterization and sequencing of the proteins
• Mass spectrometry (MS), with its low sample requirement and
high sensitivity, has been predominantly used in glycobiology for
characterization and elucidation of glycan structures
51. Disadvantages
• This often fails to distinguish between optical and geometrical isomers and
the positions of substituent in o-, m- and p- position in an aromatic ring
• Also, its scope is limited in identifying hydrocarbons that produce similar
fragmented ions.
52. Overview of MALDI-ToF
• MALDI-ToF is an instrument used to find molecular mass of a molecular substance through laser
desrption and ionization.
• Analyzes large compounds such as:
• Proteins
• Peptides Oligonucleotides
• Polymers
• MALDI considered a soft analytical technique source
53. Theory Behind MALDI-ToF
• Sample preparation
• Droplet is dried and form a solid substance
• Droplet is dried and forms a solid substance
• Plate is placed in instrument for analysis
• Sample is found using computer
• Sample analysis
• Laser shot at sample creating plumb
• Energy is transferred to matrix then to sample
• Ions travel through ToF chamber at different speeds
• Ions are detected by detector and sorted according to mass .
• Smaller ion are detected first because they travel faster
56. 2D Gel Electrophoresis
Two-dimensional gel electrophoresis (2-D electrophoresis) is a powerful
and widely used method for the analysis of complex protein mixtures
extracted from cells, tissues, or other biological samples.
1. This technique separate proteins in two steps, according to two
independent properties: the first-dimension is isoelectric focusing (IEF),
which separates proteins according to their isoelectric points (pI); the
second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE),
which separates proteins according to their molecular weights (MW).
2. In this way, complex mixtures consisted of thousands of different proteins
can be resolved and the relative amount of each protein can be determined.
57. Procedure
• The procedure involves placing the sample in gel with a pH gradient, and applying a potential
difference across it. In the electrical field, the protein migrates a long the pH gradient, until it
carries no overall charge. This location of the protein in the gel constitutes the apparent pI of
the protein.
• There are two alternatives methods to create the pH gradient - carrier ampholites and
immobilized pH gradient (IPG) gels. In the Maiman Institute for Proteome Research the IEF
is performed with commercial IPGs for highly reproducible results.
• The IEF is the most critical step of the 2-D electrophoresis process. The proteins must
be solubilize without charged detergents, usually in high concentrated urea solution,
reducing agents and chaotrophs. To obtain high quality data it is essential to achieve low
ionic strength conditions before the IEF it self. Since different types of samples differ in their
ion content, it is necessary to adjust the IEF buffer and the electrical profile to each type of
sample.
• The separation in the second dimension by molecular size is performed in slab SDS- PAGE.
Twelve parallel gels can be separated in a fixed temperature to minimize the separation
variations between individual gels.
58.
59. RPLC
Reverse Phase Liquid Chromatography (RPLC) is an extremely important subtechnique of HPLC employing
an important subset of the bonded phase chromatography. The technique is easily recognizable since, in
comparison to normal or straight phase techniques, it reverses the polarity of the original adsorbent as
well as the polarity of the mobile phase.
60. Until the nineteen-sixties, the separation of
'non volatile analytes' was often performed by
paper, and thin-layer chromatography. These
techniques were:
● Slow,
● Lacked sufficient separation power,
● Did not quantitate reliably.
History
new theoretical insights accompanied by important
developments in column packing technology and
chromatographic equipment paved the way for
what is now called High Performance or High
Pressure Liquid Chromatography (HPLC). The new
technique provided:
● Much higher resolution,
● More accurate quantitative results,
● Shorter analysis times in comparison to the
earlier techniques.
Since its introduction HPLC has evolved into an
indispensable tool in many analytical laboratories
and is applied to diverse analytical problems.
Given the advantages and its high separation
potential, RPLC has become (with the exception of
large molecules) the separation mode of choice for
the often simultaneous separation of nonpolar,
polar, and ionic analytes. Not surprisingly,
therefore, RPLC is used in a large and still growing
number of application fields. It has become an
indispensable analytical technique in many areas
61. Introduction
RPLC is the dominant analytical and preparative technique in many other scientific and
industrial settings as well. The technique is still developing, in spite of its 40+ years of use,
It employs a nonpolar stationary phase (most frequently a hydrocarbon chain chemically
bonded to porous silica particles) and a polar mobile phase constituted by water and at
least a water-miscible organic solvent, which performs as a modifier. RPLC with alkyl-
bonded stationary phases is an extraordinary separation technique that has become the
most popular HPLC mode, representing the vast majority of all HPLC separations
One of the primary factors responsible for the development of RPLC was the need of
separating mixtures containing compounds that were not sufficiently volatile or that lacked
the necessary thermal stability to be analyzed by gas chromatography, such as many polar
and ionic organic and inorganic compounds, drugs, and biomolecules.
62. Normal phase V/s Reversed phase
Reverse Phase Liquid Chromatography (RPLC) is an extremely important subtechnique of HPLC employing
an important subset of Bonded Phase Chromatography. The technique is easily recognizable since, in
comparison to normal or straight phase techniques, it reverses the polarity of the original adsorbent as
well as the polarity of the mobile phase compared to
Its flexibility and applicability to the separation of nearly all types of analytes have contributed to the
widespread use and popularity of RPLC. The following table summarizes a number of the properties of
straight and reversed phase chromatography in order to highlight the differences.
63. Advantages of RPLC
● RPLC can separate nearly all molecules with the exception of large molecules.
● A large number of high quality and efficient RPLC stationary phases are available
Combined with the wide spectrum of potential mobile phase mixtures, this offers a
range of selectivity adequate to solve nearly any separation problem.
● Water frequently forms an inexpensive, non-toxic and major part of the mobile phase.
In addition, many samples to be analyzed are soluble in aqueous-organic mixtures.
● once a suitable column has been selected, the absolute retention and selectivity
(retention relative to other solutes) can be easily manipulated using several
experimental factors, such as the percentage of the organic modifier and the
concentration of a wide range of additives (such as ion pairing reagents, surfactants,
ionic liquids, and chiral selectors), pH, and temperature.
● the easy implementation of gradient elution
● the compatibility with aqueous samples
● the possibility of separating nonpolar, polar, and ionic compounds in the same run;
64. Stationary phase
Nonpolar modified stationary phases are by far the most
important column packings in RPLC. In the majority of such
phases, alkanes or other non polar functional ligands are
covalently bonded to a support material or substrate. The
length of the carbon chains bonded to a substrate is typically
somewhere between C1 and C22. Among these stationary
phases, octyl (C-8) and octadecyl (C-18) modified silicas are
well-known and are often used as standard column packings.
Columns and stationary phases for RPLC must provide:
1. Retention and selectivity sufficient to solve a specific
analytical problem.
2. High efficiency in order to achieve acceptable
resolution
3. Satisfactory column longevity resulting from adequate
chemical, mechanical and thermal stability.
4. Excellent batch to batch and column to column
reproducibility and long term availability.
5. Acceptable pressure drop
Mobile Phase
Many RPLC protocols use a blend of water and a
miscible organic solvent (e.g., acetonitrile or
methanol) as the mobile phase. The purpose of
the organic solvent is to maintain the polarity at a
low enough level for the solute to dissolve in the
mobile phase and yet high enough to facilitate
the binding of the preferred molecule with the
stationary matrix. In some scenarios, ion-pairing
agents such as trifluoroacetic acid may also be
added. Once the molecule of interest is bound to
the column matrix, it is made to dissociate from it
by decreasing the polarity further by increasing
the concentration of the organic solvent in the
mobile phase.
This process of varying the amount of organic
solvent in the mobile phase to separate a
molecule of interest is called a gradient elution.
65. Substrate types
RPLC stationary phases can be manufactured from different susbtrate types, for example:
inorganic oxides such as silica, alumina, zirconia; or organic polymers like styrene-divinylbenzene,
methacrylates and graphitized carbons. In general, substrates for RPLC phases should meet the
following major requirements:
1. High mechanical strength adequate to withstand pressures of 500 bar and higher.
2. Uniformly shaped pores with a narrow size distribution (Ängstrom range) and sufficient
porosity,
3. Large and homogeneous surface area
4. Uniformly shaped particles (μm range) with a narrow particle size distribution.
5. No swelling or shrinking upon exposure to solvents.
6. Chemical and thermal stability under different experimental conditions.
7. High purity; free of metals contamination.
Silica meets nearly all of these requirements and, in fact, is a dream substrate for the synthesis of
RPLC phases. Silica falls short only in terms of chemical stability since it begins to dissolve at a pH
of approximately 7.5 . New synthesis and modification techniques, however, have resulted in
greatly improved silica substrates and stationary phases thereof showing high chemical and
thermal stability.
66. Silica can be produced with many different morphologies, porosities
and surface areas. For example, silica can be manufactured:
● In well defined irregularly or spherically shaped particles of
specific sizes and of controlled porosities and pore diameters.
● In addition to these more common morphologies, silica is also
widely used for the manufacture of monolithic, non- porous and
pellicular RPLC stationary phases.
The particle size of the beads is based on the specific requirements of
the separation. Larger bead size implies larger capacities and
potentially lesser pressures. Large-scale preparative processes benefit
by using beads of diameter greater than 10 μm; small-scale preparative
and analytical separations, on the other hand, benefit with beads sizes
in the 3–5 μm range.
The choice of ligand is often governed by the principle that the greater
the hydrophobicity of the molecule to be purified, the less is the need
for the ligand to be hydrophobic. This principle has also led to the rule
that chemically synthesized peptides and oligonucleotides are best
separated with C18 ligands and that protein and recombinant peptides
are better separated with C8 ligands.
67. Reference
• Ahuja, SK., Ferreira, GM. & Moreira, AR. (2004). Utilization of enzymes for
environmental applications. Critical Reviews in Biotechnology, Vol.24, No.2-3,
pp.125-154, ISSN: 0738-8551
• Akoh, CC., Chang, SW., Lee, GC. & Shaw, JF. (2008). Biocatalysis for the
production of industrial products and functional foods from rice and other
agricultural produce.
• Journal of Agricultural and Food Chemistry, Vol.56, No.22, (November 2008),
pp.10445-10451, ISSN: 0021-8561