Chapter 3(part2) - Protein purification and analysis
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Protein Purification
This document provides an overview of various protein purification techniques, including:
- Chromatography methods like affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography.
- Key steps in protein purification like cell lysis, centrifugation, assaying fractions for protein and activity, and monitoring purity with SDS-PAGE gels.
- Considerations for each chromatography method like matrix selection, binding capacities, and driving forces.
- Emerging techniques like capillary electrochromatography and multi-dimensional separations.
Protein Purification Hjp
The document provides information on protein purification techniques including:
1) Primary techniques involve breaking open cells, fractionating proteins based on size or charge, and salting out using ammonium sulfate.
2) Chromatography techniques separate proteins based on properties like binding interactions, charge, size, and isoelectric point using columns.
3) The final purity required depends on the application, with 95-99% purity needed for assays and over 99% for therapeutic use. Analytical techniques confirm identity and purity of the purified protein.
Protein Purification
The document summarizes the purification of lactate dehydrogenase (LDH) from chicken muscle. Key steps included:
1. Homogenizing the muscle tissue to disrupt cells and release LDH.
2. Removing debris via centrifugation.
3. Precipitating and concentrating LDH using ammonium sulfate.
4. Dialyzing the sample to remove salt.
5. Purifying LDH using affinity chromatography on a Cibacron blue column.
6. Analyzing purity via SDS-PAGE gel electrophoresis and activity assays.
Protein microarrays, ICAT, and HPLC protein purification
The document discusses the Isotope-Coded Affinity Tag (ICAT) method for protein quantification and identification. ICAT uses chemical labeling reagents that specifically label cysteine residues. There are 4 main steps: 1) Lyse and label protein samples from two states with light and heavy ICAT tags, 2) Mix and proteolyze samples to generate peptide fragments, some tagged, 3) Isolate tagged fragments using avidin affinity chromatography, 4) Analyze isolated peptides using mass spectrometry to identify and quantify proteins between the two states. ICAT allows accurate quantification of complex protein mixtures.
Protein purification 2008
This document provides information about an upcoming exam on protein purification techniques and related topics. It outlines the skills and concepts students should have learned, including using pipettes, spectrophotometers, organizing protocols, and preparing reports. It notes an upcoming protein purification lab experiment and encourages students to pay attention to details and properly label all samples to avoid issues. Finally, it provides an overview of the protein purification strategy and techniques to be used, including ammonium sulfate precipitation, dialysis, and column chromatography.
Protein purification
1. The document provides guidance for a tutorial on protein purification techniques for 2nd year biochemistry students.
2. Students are assigned to present on topics related to common purification methods and develop a proposed purification strategy for a specific protein.
3. An overview is given of general considerations for choosing a purification strategy based on a protein's characteristics and useful tools for primary sequence analysis. Chromatography and other methods are discussed at a high level.
Immobilized Metal affinity-chromatography
A type of Affinity chromatography which is widely used for primary purification of Histidine tagged recombinant proteins
Campbell6e lecture ch5
This document summarizes several common techniques used to purify and characterize proteins:
1. Proteins are first isolated from cells through homogenization and then separated from contaminants using techniques like salting out with ammonium sulfate or differential centrifugation.
2. Column chromatography techniques like size-exclusion chromatography, affinity chromatography, and ion exchange chromatography are used to further purify proteins based on properties like size, specific binding interactions, or charge.
3. Electrophoresis, isoelectric focusing, and techniques like amino acid analysis, Edman degradation, and enzymatic or chemical cleavage are then used to determine a protein's primary structure by separating peptide fragments and determining their sequence.
Recommended
Protein Purification
This document provides an overview of various protein purification techniques, including:
- Chromatography methods like affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography.
- Key steps in protein purification like cell lysis, centrifugation, assaying fractions for protein and activity, and monitoring purity with SDS-PAGE gels.
- Considerations for each chromatography method like matrix selection, binding capacities, and driving forces.
- Emerging techniques like capillary electrochromatography and multi-dimensional separations.
Protein Purification Hjp
The document provides information on protein purification techniques including:
1) Primary techniques involve breaking open cells, fractionating proteins based on size or charge, and salting out using ammonium sulfate.
2) Chromatography techniques separate proteins based on properties like binding interactions, charge, size, and isoelectric point using columns.
3) The final purity required depends on the application, with 95-99% purity needed for assays and over 99% for therapeutic use. Analytical techniques confirm identity and purity of the purified protein.
Protein Purification
The document summarizes the purification of lactate dehydrogenase (LDH) from chicken muscle. Key steps included:
1. Homogenizing the muscle tissue to disrupt cells and release LDH.
2. Removing debris via centrifugation.
3. Precipitating and concentrating LDH using ammonium sulfate.
4. Dialyzing the sample to remove salt.
5. Purifying LDH using affinity chromatography on a Cibacron blue column.
6. Analyzing purity via SDS-PAGE gel electrophoresis and activity assays.
Protein microarrays, ICAT, and HPLC protein purification
The document discusses the Isotope-Coded Affinity Tag (ICAT) method for protein quantification and identification. ICAT uses chemical labeling reagents that specifically label cysteine residues. There are 4 main steps: 1) Lyse and label protein samples from two states with light and heavy ICAT tags, 2) Mix and proteolyze samples to generate peptide fragments, some tagged, 3) Isolate tagged fragments using avidin affinity chromatography, 4) Analyze isolated peptides using mass spectrometry to identify and quantify proteins between the two states. ICAT allows accurate quantification of complex protein mixtures.
Protein purification 2008
This document provides information about an upcoming exam on protein purification techniques and related topics. It outlines the skills and concepts students should have learned, including using pipettes, spectrophotometers, organizing protocols, and preparing reports. It notes an upcoming protein purification lab experiment and encourages students to pay attention to details and properly label all samples to avoid issues. Finally, it provides an overview of the protein purification strategy and techniques to be used, including ammonium sulfate precipitation, dialysis, and column chromatography.
Protein purification
1. The document provides guidance for a tutorial on protein purification techniques for 2nd year biochemistry students.
2. Students are assigned to present on topics related to common purification methods and develop a proposed purification strategy for a specific protein.
3. An overview is given of general considerations for choosing a purification strategy based on a protein's characteristics and useful tools for primary sequence analysis. Chromatography and other methods are discussed at a high level.
Immobilized Metal affinity-chromatography
A type of Affinity chromatography which is widely used for primary purification of Histidine tagged recombinant proteins
Campbell6e lecture ch5
This document summarizes several common techniques used to purify and characterize proteins:
1. Proteins are first isolated from cells through homogenization and then separated from contaminants using techniques like salting out with ammonium sulfate or differential centrifugation.
2. Column chromatography techniques like size-exclusion chromatography, affinity chromatography, and ion exchange chromatography are used to further purify proteins based on properties like size, specific binding interactions, or charge.
3. Electrophoresis, isoelectric focusing, and techniques like amino acid analysis, Edman degradation, and enzymatic or chemical cleavage are then used to determine a protein's primary structure by separating peptide fragments and determining their sequence.
Protein purification
The document discusses protein purification, including determining goals such as protein yield and purity needs. It describes preparing proteins through natural sources or recombinant expression, which may involve tags, mutations, or codon optimization. The purification process typically involves multiple chromatography steps such as affinity, ion exchange, and size exclusion to capture the protein from cell extracts and progressively enrich and polish it to the desired purity, with yields decreasing at each step. Expression systems, hosts, promoters, and tags can be chosen to optimize solubility and purification. Protease inhibitors and quick processing help maintain protein stability.
Analytical techniques for separation or purification of proteins
A comprehensive presentation on Analytical techniques for separation or purification of proteins for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.
Downstream Processing in Biopharmaceuticals
This document discusses downstream processing in biopharmaceuticals. It provides examples of characterizing proteins like human serum albumin, describing its molecular weight, isoelectric point, and hydropathicity. It also discusses large scale protein production methods like transfection, purification using affinity and size exclusion chromatography, and challenges like glycosylation that can be addressed through techniques such as mutagenesis and using insect cells. The typical downstream processing flow is outlined involving clarification, filtration, chromatography, and viral clearance steps.
chromatofocusing, 2 de, ief
Chromatofocusing is a protein separation technique that uses ion exchange resins and buffers with changing pH to separate proteins based on their isoelectric point. As the buffer pH passes through a protein's pI, the protein loses its charge and elutes from the resin. Chromatofocusing provides high resolution separation of proteins that have similar pI values. However, some proteins may aggregate at high concentrations and clog the resin. Isoelectric focusing uses immobilized pH gradients in gels to separate proteins based on their pI through electrophoresis. Two-dimensional electrophoresis separates proteins first by pI using isoelectric focusing, then by molecular weight using SDS-PAGE to provide high resolution separation and identification of
Protein separation
The document summarizes strategies for protein purification. It discusses that protein purification separates and isolates proteins from complex mixtures using differences in their physical and chemical properties. It outlines various centrifugation and chromatography techniques used in protein purification, including differential centrifugation, gel filtration, ion exchange chromatography, and affinity chromatography. These techniques separate proteins based on properties like size, charge, and binding affinity. The document also notes that protein purification is now performed from research to industrial scales and that affinity tagging has revolutionized the field.
Protein Electrophoresis & Gas Liquid Chromatography & HPLC Applications
This document provides information on several chromatography techniques including serum protein electrophoresis, gas chromatography, and high performance liquid chromatography. It describes the basic procedures, clinical applications, and significance of each method. Serum protein electrophoresis is used to separate and quantify protein fractions in serum to diagnose conditions such as hypoalbuminemia and multiple myeloma. Gas chromatography separates volatile compounds and is applied in forensics to analyze bodily fluids. High performance liquid chromatography is widely used to separate non-volatile compounds such as proteins, nucleic acids, and pharmaceuticals.
Antibody purification – what you need to know to use antibodies effectively
In this webinar Dr Andy Lane discusses the various methods available for purifying antibodies from different sources, and explains why it is vitally important to understand how your antibodies have been purified to know what you can do with them, either within assays or for further processing such as conjugation to dyes and enzymes.
Global analysis of protein
Proteins play crucial roles in nearly all biological processes. These many functions of proteins are a result of the folding of proteins into many distinct 3D structures.
Protein analysis tries to explore how amino acid sequences specify the structure of proteins and how these proteins bind to substrates and other molecules to perform their functions.
Protein analysis allows us to understand the function of the protein based on its structure.
Protein analysis
The document summarizes different methods for protein analysis, including qualitative and quantitative techniques. It discusses the history of protein analysis and introduces various methods such as the Biuret test, spectroscopy, chromatography, and electrophoresis. Specific techniques are described in detail, such as ion exchange chromatography, affinity chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The document concludes by discussing size exclusion chromatography and provides references on the topic of protein analysis.
Purification techniques chromatography
This document discusses various chromatography techniques used to purify biomolecules, including affinity chromatography, ion exchange chromatography, size exclusion chromatography, and reversed phase chromatography. It provides details on how each technique separates molecules based on specific properties, such as biological recognition (affinity chromatography), charge (ion exchange chromatography), size (size exclusion chromatography), and hydrophobicity (reversed phase chromatography).
Protein Purification Lecture
This document provides guidance on protein purification methods. It discusses lysing cells to release the target protein, protecting the protein from degradation during purification, tracking the protein using activity assays and purification tables, initial gentle fractionation using ammonium sulfate precipitation, chromatographic separation methods like ion exchange and affinity chromatography, and final polishing steps like additional chromatography or ultrafiltration. The overall goal is to leverage the target protein's unique properties to separate it from contaminants through a series of purification techniques.
Purification techniques
Protein purification techniques can be categorized into those based on molecular size, solubility, and electric charge. Size-based techniques include dialysis, ultrafiltration, and size-exclusion chromatography which separate proteins based on their ability to pass through semi-permeable membranes or porous beads. Solubility-based techniques include isoelectric precipitation and salting out which alter a protein's solubility by adjusting pH or salt concentration. Charge-based techniques such as ion-exchange and electrophoresis separate proteins using their net electric charge in an applied electric field or ion-exchange column.
Protein purification chp-5-bioc-361-version-oct-2012
very simple view of protein purification which is a small component of the course here (chem 361). Mostly from Campbell 6th ed. with a small bit added on 2D gels.
Characterization of protein
Introduction
Proteins
Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis
Steps Of Protein Characterisation:
Determination Of Protein Concentration
Biuret Reaction
Lowry (Folin-Lowry) Method
UV- Spectroscopy
Assessment Of Protein Purity
SDS -Phage
Immunoblot
Surface Charge Analysis
Isoelectro Focusing
Ion Exchange Chromatography
Size, Shape And Conformation Analysis
2d-Electrophorasis
X-Ray Crytalliography
Protein Structure and Sequence Analysis
Edman Sequencing
Conclusion
References
10.analytical biochemistry
This document discusses analytical biochemistry techniques for isolating and sequencing proteins. It covers cell disruption methods like sonication, centrifugation types including differential and density gradient, and spectrophotometry. Cell disruption is needed to extract proteins from cells. Centrifugation separates particles based on size, shape and density. Differential centrifugation separates with increasing g-force while density gradient centrifugation uses a medium with increasing density. Spectrophotometry analyzes light absorption of substances using Lambert's and Beer's laws.
Extraction buffer, Protease inhibitors methods of cell distrubtion
This document discusses methods for extracting and preparing proteins for proteomics analysis. It describes how to prepare stable buffer solutions to maintain pH during extraction. Protease inhibitors are also important to prevent proteolysis during extraction and purification. Various mechanical and enzymatic cell lysis techniques are covered, such as sonication, French press, and enzymatic lysis of yeast. Considerations for extracting from prokaryotes, eukaryotes, and animal tissues are provided. Centrifugation can be used to separate subcellular fractions after homogenization.
Protein Detection Methods and Application
The document discusses various protein analysis methods including gel electrophoresis, SDS-PAGE, and native gel electrophoresis. Gel electrophoresis separates proteins, DNA, and RNA using an electric current applied to a gel matrix. SDS-PAGE separates denatured proteins by size, coating them with negative charges. Native gel electrophoresis separates intact proteins by their intrinsic charge and hydrodynamic size, allowing analysis of conformation, aggregation, and binding events.
Exploring Proteins and Proteomes. Stryer,CHAPTER 3 ppt
Methods in Protein Chemistry
This chapter discusses several methods used to isolate, purify, detect, degrade, analyze, and synthesize proteins. It describes techniques such as centrifugation, solubility, dialysis, gel filtration, affinity chromatography, HPLC, electrophoresis, and mass spectrometry. It also covers determining a protein's amino acid sequence through methods like Edman degradation, solid phase synthesis, chemical and enzymatic cleavage, and the use of DNA sequencing. The goal of these methods is to obtain a protein's amino acid sequence and gain functional information about proteins and proteomes.
Characterization of proteins
The document discusses various techniques for protein purification and characterization, including:
1. Detergents solubilize transmembrane proteins by having affinity for hydrophobic groups and water.
2. Centrifugation separates particles of different masses or densities, with denser particles pelleted first.
3. Electrophoresis separates charged particles in an electrical field depending on their charge and size.
4. Chromatography techniques separate proteins based on properties like size, charge, or binding affinity.
Affinity chromatography and gel filteration
Affinity chromatography is a technique that uses the specific binding properties of a molecule to purify it from a mixed sample. The process involves passing a sample over a column containing beads with affinity ligands attached. Proteins in the sample either bind tightly, bind loosely, or do not bind to the ligands on the beads. Multiple wash steps are used to remove non-binding and loosely binding proteins, leaving only the tightly bound protein of interest, which can then be eluted and collected in a purified form.
the cell
This document provides an overview and summaries of chapter 6 from Campbell Biology, 9th edition, which discusses a tour of the cell. The chapter introduces cells as the fundamental unit of life and explains how cell structure relates to function. It describes how biologists use microscopes like light microscopes and electron microscopes as well as techniques like cell fractionation to study cells. The document includes figures illustrating the size scales visible with different microscopes and an illustration of the cell fractionation technique.
Chaperone - Molecular Biology
The document summarizes two studies: 1) A study found that the plasma protein clusterin is associated with hippocampal atrophy and rapid cognitive decline in Alzheimer's disease. This could lead to treatments that stop disease progression. 2) A separate study found that increasing levels of heat shock protein Hsp70 through inhibition of Hsp90 reversed nerve damage in diabetic mice. This treatment approach could lead to new medicines for diabetic patients. Overall, the studies provide insight into disease mechanisms and potential new treatment strategies for Alzheimer's disease and diabetes.
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Protein purification
The document discusses protein purification, including determining goals such as protein yield and purity needs. It describes preparing proteins through natural sources or recombinant expression, which may involve tags, mutations, or codon optimization. The purification process typically involves multiple chromatography steps such as affinity, ion exchange, and size exclusion to capture the protein from cell extracts and progressively enrich and polish it to the desired purity, with yields decreasing at each step. Expression systems, hosts, promoters, and tags can be chosen to optimize solubility and purification. Protease inhibitors and quick processing help maintain protein stability.
Analytical techniques for separation or purification of proteins
A comprehensive presentation on Analytical techniques for separation or purification of proteins for MBBS , BDS, B Pharm & Biotechnology students to facilitate self- study.
Downstream Processing in Biopharmaceuticals
This document discusses downstream processing in biopharmaceuticals. It provides examples of characterizing proteins like human serum albumin, describing its molecular weight, isoelectric point, and hydropathicity. It also discusses large scale protein production methods like transfection, purification using affinity and size exclusion chromatography, and challenges like glycosylation that can be addressed through techniques such as mutagenesis and using insect cells. The typical downstream processing flow is outlined involving clarification, filtration, chromatography, and viral clearance steps.
chromatofocusing, 2 de, ief
Chromatofocusing is a protein separation technique that uses ion exchange resins and buffers with changing pH to separate proteins based on their isoelectric point. As the buffer pH passes through a protein's pI, the protein loses its charge and elutes from the resin. Chromatofocusing provides high resolution separation of proteins that have similar pI values. However, some proteins may aggregate at high concentrations and clog the resin. Isoelectric focusing uses immobilized pH gradients in gels to separate proteins based on their pI through electrophoresis. Two-dimensional electrophoresis separates proteins first by pI using isoelectric focusing, then by molecular weight using SDS-PAGE to provide high resolution separation and identification of
Protein separation
The document summarizes strategies for protein purification. It discusses that protein purification separates and isolates proteins from complex mixtures using differences in their physical and chemical properties. It outlines various centrifugation and chromatography techniques used in protein purification, including differential centrifugation, gel filtration, ion exchange chromatography, and affinity chromatography. These techniques separate proteins based on properties like size, charge, and binding affinity. The document also notes that protein purification is now performed from research to industrial scales and that affinity tagging has revolutionized the field.
Protein Electrophoresis & Gas Liquid Chromatography & HPLC Applications
This document provides information on several chromatography techniques including serum protein electrophoresis, gas chromatography, and high performance liquid chromatography. It describes the basic procedures, clinical applications, and significance of each method. Serum protein electrophoresis is used to separate and quantify protein fractions in serum to diagnose conditions such as hypoalbuminemia and multiple myeloma. Gas chromatography separates volatile compounds and is applied in forensics to analyze bodily fluids. High performance liquid chromatography is widely used to separate non-volatile compounds such as proteins, nucleic acids, and pharmaceuticals.
Antibody purification – what you need to know to use antibodies effectively
In this webinar Dr Andy Lane discusses the various methods available for purifying antibodies from different sources, and explains why it is vitally important to understand how your antibodies have been purified to know what you can do with them, either within assays or for further processing such as conjugation to dyes and enzymes.
Global analysis of protein
Proteins play crucial roles in nearly all biological processes. These many functions of proteins are a result of the folding of proteins into many distinct 3D structures.
Protein analysis tries to explore how amino acid sequences specify the structure of proteins and how these proteins bind to substrates and other molecules to perform their functions.
Protein analysis allows us to understand the function of the protein based on its structure.
Protein analysis
The document summarizes different methods for protein analysis, including qualitative and quantitative techniques. It discusses the history of protein analysis and introduces various methods such as the Biuret test, spectroscopy, chromatography, and electrophoresis. Specific techniques are described in detail, such as ion exchange chromatography, affinity chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The document concludes by discussing size exclusion chromatography and provides references on the topic of protein analysis.
Purification techniques chromatography
This document discusses various chromatography techniques used to purify biomolecules, including affinity chromatography, ion exchange chromatography, size exclusion chromatography, and reversed phase chromatography. It provides details on how each technique separates molecules based on specific properties, such as biological recognition (affinity chromatography), charge (ion exchange chromatography), size (size exclusion chromatography), and hydrophobicity (reversed phase chromatography).
Protein Purification Lecture
This document provides guidance on protein purification methods. It discusses lysing cells to release the target protein, protecting the protein from degradation during purification, tracking the protein using activity assays and purification tables, initial gentle fractionation using ammonium sulfate precipitation, chromatographic separation methods like ion exchange and affinity chromatography, and final polishing steps like additional chromatography or ultrafiltration. The overall goal is to leverage the target protein's unique properties to separate it from contaminants through a series of purification techniques.
Purification techniques
Protein purification techniques can be categorized into those based on molecular size, solubility, and electric charge. Size-based techniques include dialysis, ultrafiltration, and size-exclusion chromatography which separate proteins based on their ability to pass through semi-permeable membranes or porous beads. Solubility-based techniques include isoelectric precipitation and salting out which alter a protein's solubility by adjusting pH or salt concentration. Charge-based techniques such as ion-exchange and electrophoresis separate proteins using their net electric charge in an applied electric field or ion-exchange column.
Protein purification chp-5-bioc-361-version-oct-2012
very simple view of protein purification which is a small component of the course here (chem 361). Mostly from Campbell 6th ed. with a small bit added on 2D gels.
Characterization of protein
Introduction
Proteins
Function Of Protein And Their Properties
Protein Isolation And Purification
Methods Of Cell Lysis
Steps Of Protein Characterisation:
Determination Of Protein Concentration
Biuret Reaction
Lowry (Folin-Lowry) Method
UV- Spectroscopy
Assessment Of Protein Purity
SDS -Phage
Immunoblot
Surface Charge Analysis
Isoelectro Focusing
Ion Exchange Chromatography
Size, Shape And Conformation Analysis
2d-Electrophorasis
X-Ray Crytalliography
Protein Structure and Sequence Analysis
Edman Sequencing
Conclusion
References
10.analytical biochemistry
This document discusses analytical biochemistry techniques for isolating and sequencing proteins. It covers cell disruption methods like sonication, centrifugation types including differential and density gradient, and spectrophotometry. Cell disruption is needed to extract proteins from cells. Centrifugation separates particles based on size, shape and density. Differential centrifugation separates with increasing g-force while density gradient centrifugation uses a medium with increasing density. Spectrophotometry analyzes light absorption of substances using Lambert's and Beer's laws.
Extraction buffer, Protease inhibitors methods of cell distrubtion
This document discusses methods for extracting and preparing proteins for proteomics analysis. It describes how to prepare stable buffer solutions to maintain pH during extraction. Protease inhibitors are also important to prevent proteolysis during extraction and purification. Various mechanical and enzymatic cell lysis techniques are covered, such as sonication, French press, and enzymatic lysis of yeast. Considerations for extracting from prokaryotes, eukaryotes, and animal tissues are provided. Centrifugation can be used to separate subcellular fractions after homogenization.
Protein Detection Methods and Application
The document discusses various protein analysis methods including gel electrophoresis, SDS-PAGE, and native gel electrophoresis. Gel electrophoresis separates proteins, DNA, and RNA using an electric current applied to a gel matrix. SDS-PAGE separates denatured proteins by size, coating them with negative charges. Native gel electrophoresis separates intact proteins by their intrinsic charge and hydrodynamic size, allowing analysis of conformation, aggregation, and binding events.
Exploring Proteins and Proteomes. Stryer,CHAPTER 3 ppt
Methods in Protein Chemistry
This chapter discusses several methods used to isolate, purify, detect, degrade, analyze, and synthesize proteins. It describes techniques such as centrifugation, solubility, dialysis, gel filtration, affinity chromatography, HPLC, electrophoresis, and mass spectrometry. It also covers determining a protein's amino acid sequence through methods like Edman degradation, solid phase synthesis, chemical and enzymatic cleavage, and the use of DNA sequencing. The goal of these methods is to obtain a protein's amino acid sequence and gain functional information about proteins and proteomes.
Characterization of proteins
The document discusses various techniques for protein purification and characterization, including:
1. Detergents solubilize transmembrane proteins by having affinity for hydrophobic groups and water.
2. Centrifugation separates particles of different masses or densities, with denser particles pelleted first.
3. Electrophoresis separates charged particles in an electrical field depending on their charge and size.
4. Chromatography techniques separate proteins based on properties like size, charge, or binding affinity.
Affinity chromatography and gel filteration
Affinity chromatography is a technique that uses the specific binding properties of a molecule to purify it from a mixed sample. The process involves passing a sample over a column containing beads with affinity ligands attached. Proteins in the sample either bind tightly, bind loosely, or do not bind to the ligands on the beads. Multiple wash steps are used to remove non-binding and loosely binding proteins, leaving only the tightly bound protein of interest, which can then be eluted and collected in a purified form.
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Chaperone - Molecular Biology
The document summarizes two studies: 1) A study found that the plasma protein clusterin is associated with hippocampal atrophy and rapid cognitive decline in Alzheimer's disease. This could lead to treatments that stop disease progression. 2) A separate study found that increasing levels of heat shock protein Hsp70 through inhibition of Hsp90 reversed nerve damage in diabetic mice. This treatment approach could lead to new medicines for diabetic patients. Overall, the studies provide insight into disease mechanisms and potential new treatment strategies for Alzheimer's disease and diabetes.
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This document discusses various methods for determining the amino acid sequence of proteins, including:
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HEME DEGRADATION
Erythrocytes have a lifespan of 120 days before being removed from circulation and degraded by macrophages in the spleen and liver. Approximately 6 grams of hemoglobin is broken down per day in an adult human. Heme is broken down by the enzyme heme oxygenase into biliverdin, iron, and carbon monoxide. Biliverdin is further degraded by biliverdin reductase into bilirubin, which is transported bound to albumin in plasma and taken up by hepatocytes in the liver. In the liver, bilirubin is conjugated and excreted into bile as bilirubin diglucuronide.
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1. The document outlines common strategies used for protein sequencing, including Edman degradation. Edman degradation involves breaking the peptide bonds of a protein one by one to determine the amino acid sequence.
2. Other strategies discussed include peptide mass fingerprinting, tandem mass spectrometry, and de novo sequencing methods. Peptide mass fingerprinting involves breaking a protein into peptides and using mass spectrometry to determine the peptide masses and compare them to databases. Tandem mass spectrometry further breaks peptides into fragment ions for sequencing.
3. The strategies each have advantages and limitations. While Edman degradation was previously standard, mass spectrometry techniques are increasingly used due to their high-throughput capabilities and ability to sequence smaller protein amounts. The
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Amino acid sequencing
Amino acid sequencing determines the order of amino acids in a protein. Frederick Sanger determined the first protein sequence in 1953 using N-terminal analysis methods like Edman degradation. Large proteins are sequenced by first breaking them into smaller fragments using enzymes or chemicals, then determining the sequence of individual fragments and combining sequences to deduce the full protein sequence. Modern techniques like mass spectrometry have made sequencing faster and applicable to modified proteins.
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- 3. Steps in protein purification • Develop assay • Choose source of protein • Prepare tissue extract – cell disruption – subcellular fractionation • Protein fractionation (several steps) • Determination of purity
- 4. Differential Centrifugation 1000 g tissue homogenate transfer supernatant Pellet unbroken cells nuclei chloroplast transfer supernatant transfer supernatant 10,000 g 100,000 g Pellet mitochondria Pellet microsomal Fraction (ER, golgi, lysosomes, peroxisomes) Super. Cytosol, Soluble enzymes
- 7. Ion-exchange Chromatography - - - - - - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - ++++++ ++++++ ++++++ low salt buffer high salt buffer + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Cl- + + + + + + + + + + - - - - - - - - - Cl- Cl- Cl- Cl- Cl- - - - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - - - - - ++++++ ++++++ ++++++
- 8. Affinity Chromatography Add excess ligand
- 9. SDS poly acrylamide electrophoresis (PAGE) SDS = H3C-(CH2)10-CH2-OSO3 - SDS – denatures protein coats w/ negative charge - - - - - - - - - - - - - -- - Used to determine protein MW And purity of protein prep
- 10. Isoelectric Focusing pH 9 - - Decreasing pH + Decreasing pH + pH 3
- 11. 2-D Electrophoresis Decreasing MW large small Decreasing pH - + SDS-PAGE Decreasing pH Decreasing MW
- 12. Amino Acid Analysis H N C S H3N C O- N O C R S C HN C C H O R 1) Acid hydrolyze protein 2) Treat with phenylisothiocyanate (PICT) + 3) Separate derivatized AA’s by HPLC
- 13. Protein Sequencing (Edman Degradation) H N C S H3N C O C R H NH C O C R X S N C H HN C O C R H NH C O C R X + Trifluoroacetic acid N S C HN C C H O R H 2HN C O C R X 1) 2) 3) Repeat Can sequence in 30 to 60 AA’s from N-terminus
- 14. Generate Proteolytic Fragments Endopeptidases •Typsin cleaves at COOH end of Lys and Arg •Chymotrypsin cleaves at COOH end of Phe, Tyr, Trp Chemical Cleavages •Cyanogen Bromide cleaves at COOH end of Met Generate overlapping fragments Sequence individual fragments and piece together sequence
- 15. Peptide mapping exercise Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp Trypsin Met-Ala-Arg Phe-Ala-Glu-Gln-Asp Gly-Glu-Tyr-Met-Cys-Lys Chymotrysin Met-Ala-Arg- Gly-Glu-Tyr Met-Cys-Lys –Phe Ala-Glu-Gln-Asp CNBr Met Ala-Arg-Gly-Glu-Tyr-Met Cys-Lys-Phe-Ala-Glu-Gln-Asp
- 17. Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF)