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The document outlines the various steps involved in purifying an enzyme, including: 1) Removal of nucleic acids and cell debris through centrifugation, filtration, or precipitation with chemicals like protamine sulfate or streptomycin. 2) Preliminary purification steps to remove small molecules and unwanted contaminants. 3) Separation based on solubility differences, often using precipitation mediated by changes in salt concentration, pH, or temperature. 4) Final purification techniques like chromatography, adsorption, or concentration to achieve a highly purified enzyme.
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Techniques for protein purification
Protein purification involves a series of steps to isolate a single protein from a complex mixture. These steps may separate proteins based on size, charge, or binding affinity. Common techniques include precipitation with ammonium sulfate, chromatography methods like ion exchange, affinity, size exclusion, and reversed-phase chromatography, and electrophoresis. The goal is to free the protein of interest from other materials, separate it from other proteins, and finally isolate it in a pure form for characterization and use.
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This document discusses strategies for purifying proteins using chromatography techniques. It recommends using a three-phase purification strategy including capture, intermediate purification, and polishing steps. The key is selecting appropriate techniques based on the protein's properties and combining techniques in a logical sequence to maximize yield while minimizing steps. Common chromatography techniques discussed include ion exchange, size exclusion, hydrophobic interaction, and affinity chromatography.
Isolation purification technique for Therapeutic Bio molecules : Protein-Peptide
The document discusses isolation and purification techniques of therapeutic biomolecules. It describes how proteins and peptides can be isolated from animal sources like pancreas, brains, and salmon. Magnetic separation techniques like magnetic-activated cell sorting (MACS) can be used to isolate target proteins and peptides. The document also discusses various methods used for sample disruption like grinding, shearing, beating and shocking. Key techniques for purification of isolated biomolecules include ion exchange chromatography, size exclusion chromatography, ammonium sulfate precipitation, and high pressure liquid chromatography.
Protein purification stratigies
This document discusses various protein purification strategies and techniques. It outlines steps for protein isolation, extraction, concentration, and precipitation. It also describes different chromatography methods for purification, including gel filtration, ion exchange, hydrophobic interaction, and affinity chromatography. It provides details on analytical tools like SDS-PAGE for assessing purity and discusses considerations for combining purification techniques.
Protein purification techniques
The document discusses various techniques for purifying proteins. General steps in protein purification include selecting a protein source, homogenizing and solubilizing the proteins, and stabilizing the proteins. Specific techniques discussed include ammonium sulfate precipitation, dialysis, gel filtration chromatography, ion exchange chromatography, affinity chromatography, fast performance liquid chromatography, and gel electrophoresis. The goal of protein purification is to isolate a single protein of interest from other contaminating proteins in order to study its structure and function.
Protein purification
Protein purification is a series of processes to isolate a protein from a complex mixture. It is important to characterize a protein's function, structure, and interactions. The general steps of purification include preparing the source, exploiting differences in protein properties through separation techniques, and monitoring purity with assays. Purification yields a pure protein sample for determining its sequence, structure, and role. Affinity tags and other tags are often used to aid purification. Biopharmaceuticals are medically important proteins produced through biotechnology.
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 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.
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Techniques for protein purification
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Protein purification stratigies
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Protein purification
Protein purification is a series of processes to isolate a protein from a complex mixture. It is important to characterize a protein's function, structure, and interactions. The general steps of purification include preparing the source, exploiting differences in protein properties through separation techniques, and monitoring purity with assays. Purification yields a pure protein sample for determining its sequence, structure, and role. Affinity tags and other tags are often used to aid purification. Biopharmaceuticals are medically important proteins produced through biotechnology.
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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.
Lec tech.protein separation
Protein purification involves a series of processes to isolate a single type of protein from a complex mixture. The starting material is usually a biological tissue or microbial culture. Various methods are used to separate the protein from non-protein parts and finally separate the desired protein from all others. Key methods include centrifugation, precipitation, chromatography, electrophoresis, and ultrafiltration which separate proteins based on properties like size, charge, and hydrophobicity. The goal is to purify the protein of interest to characterize its structure, function, and interactions.
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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.
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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.
Affinity chromatography
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This document provides an overview of methods used to study and purify proteins. It discusses techniques such as homogenization, differential centrifugation, salting out, dialysis, and various types of chromatography that can be used to isolate proteins from cell lysates and purify them. The document emphasizes that purifying proteins is essential for understanding their structure and function through in vitro, in vivo, and in silico methods of study.
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Affinity chromatography is a separation technique that uses the specific interactions between biomolecules to selectively isolate target molecules from complex mixtures. The interacting partner of the target molecule is immobilized on a chromatographic resin as a ligand. When a sample containing the target molecule flows through the column, the target molecule will reversibly bind to the ligand. By changing the composition of the mobile phase, such as pH or ionic strength, the bound target molecule can then be eluted. Affinity chromatography provides high selectivity and yield due to the specific interactions used, and in some cases allows for single-step isolation of target molecules from samples.
Protein purification
Protein purification techniques take advantage of differences in protein properties like charge, size, and binding affinity. The first step is breaking open cells to release proteins. Centrifugation is used to separate subcellular components. Fractionation utilizes differences in solubility, often using ammonium sulfate precipitation. Dialysis removes small solutes by exchanging them through a semipermeable membrane. Column chromatography separates proteins as they migrate through a solid matrix at different rates depending on their interactions. Specific techniques further separate proteins based on ionic charge using ion-exchange, size using size exclusion, or binding affinity using affinity chromatography.
Affinity Chromatography
Affinity chromatography is a separation technique that relies on the specific binding interaction between an immobilized ligand and its binding partner. It is commonly used to purify biomolecules like proteins and enzymes. The stationary phase contains a solid support with an affinity ligand that selectively binds the target molecule. The sample is loaded and the target molecule binds while contaminants are washed away. The bound target is then eluted by changing conditions to disrupt the binding. Affinity chromatography offers high specificity and purity but can be time-consuming and require expensive ligands.
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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.
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
Affinity chromatography
Affinity chromatography is a type of liquid chromatography that uses reversible biological interactions or molecular recognition to separate sample components. It works by attaching a ligand with affinity for the target molecule to a stationary phase. When a sample mixture passes through the column, only molecules that bind to the ligand will be retained on the stationary phase while non-binding molecules pass through. Retained molecules can then be eluted by altering conditions to break the binding interaction selectively. Affinity chromatography is useful for purifying and studying enzymes, proteins, and other biomolecules due to its high specificity and ability to achieve a high degree of purity.
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This document provides an overview of various protein purification techniques, including:
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- 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.
Proteins purification
This document discusses various methods for purifying proteins, including selecting an appropriate source, lysing cells, centrifugation, chromatography, electrophoresis, and measuring protein activity. Key steps involve disrupting cells to release proteins, separating components by techniques that exploit differences in properties like size, charge, and affinity for ligands, and ensuring the purified protein retains its native structure and activity. The goal is to enrich the target protein away from other biomolecules to obtain a pure sample for downstream applications.
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Lec tech.protein separation
Protein purification involves a series of processes to isolate a single type of protein from a complex mixture. The starting material is usually a biological tissue or microbial culture. Various methods are used to separate the protein from non-protein parts and finally separate the desired protein from all others. Key methods include centrifugation, precipitation, chromatography, electrophoresis, and ultrafiltration which separate proteins based on properties like size, charge, and hydrophobicity. The goal is to purify the protein of interest to characterize its structure, function, and interactions.
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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 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.
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This document discusses protein purification techniques. It begins by outlining the purposes of protein purification, which include producing commercial products and conducting research. It then describes different methods for separating and purifying proteins based on their location in or outside of cells. These include centrifugation, salt precipitation, various types of chromatography (size exclusion, ion exchange, affinity), and electrophoresis. The key steps are cell disruption when needed followed by differential centrifugation, then one or more chromatography techniques which can achieve high levels of purity.
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.
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.
Affinity chromatography
Affinity chromatography is a purification technique used for biological molecules like proteins. It works by immobilizing a ligand with specific affinity for the target molecule on a support. When a mixture is passed through the column, the target molecule binds selectively to the ligand. Common stationary phases are agarose gels. Various affinity media exist that exploit properties like metal ion interactions, carbohydrate binding, or antigen-antibody reactions. Applications include purification of nucleic acids, proteins, antibodies, and enzymes from cell extracts in fields like genetic engineering, vaccine production, and basic research.
219158 lecture 9
This document provides an overview of methods used to study and purify proteins. It discusses techniques such as homogenization, differential centrifugation, salting out, dialysis, and various types of chromatography that can be used to isolate proteins from cell lysates and purify them. The document emphasizes that purifying proteins is essential for understanding their structure and function through in vitro, in vivo, and in silico methods of study.
Affinity Chromatography
Affinity chromatography is a separation technique that uses the specific binding interaction between an immobilized ligand and its binding partner. It relies on a solid support with an affinity ligand that selectively binds the target molecule. The target molecule is purified in a single step by binding to the ligand, washing away unbound molecules, and then eluting the target molecule under different conditions. Affinity chromatography provides high selectivity and purity and is widely used to purify proteins and other biomolecules.
Introduction to Affinity Chromatography
Affinity chromatography is a separation technique that uses the specific interactions between biomolecules to selectively isolate target molecules from complex mixtures. The interacting partner of the target molecule is immobilized on a chromatographic resin as a ligand. When a sample containing the target molecule flows through the column, the target molecule will reversibly bind to the ligand. By changing the composition of the mobile phase, such as pH or ionic strength, the bound target molecule can then be eluted. Affinity chromatography provides high selectivity and yield due to the specific interactions used, and in some cases allows for single-step isolation of target molecules from samples.
Protein purification
Protein purification techniques take advantage of differences in protein properties like charge, size, and binding affinity. The first step is breaking open cells to release proteins. Centrifugation is used to separate subcellular components. Fractionation utilizes differences in solubility, often using ammonium sulfate precipitation. Dialysis removes small solutes by exchanging them through a semipermeable membrane. Column chromatography separates proteins as they migrate through a solid matrix at different rates depending on their interactions. Specific techniques further separate proteins based on ionic charge using ion-exchange, size using size exclusion, or binding affinity using affinity chromatography.
Affinity Chromatography
Affinity chromatography is a separation technique that relies on the specific binding interaction between an immobilized ligand and its binding partner. It is commonly used to purify biomolecules like proteins and enzymes. The stationary phase contains a solid support with an affinity ligand that selectively binds the target molecule. The sample is loaded and the target molecule binds while contaminants are washed away. The bound target is then eluted by changing conditions to disrupt the binding. Affinity chromatography offers high specificity and purity but can be time-consuming and require expensive ligands.
Purification of proteins (purification of enzymes)
This document discusses the purification of proteins. It begins with an introduction to proteins and their structures. It then discusses various techniques used to purify proteins, including salting out, dialysis, gel filtration chromatography, ion-exchange chromatography, gel electrophoresis, isoelectric focusing, and HPLC. The key techniques discussed in detail are salting out, dialysis, gel filtration chromatography, ion-exchange chromatography, gel electrophoresis, and affinity chromatography. The document emphasizes that different techniques are used to purify proteins based on their properties such as solubility, molecular size, ionic charge, and binding specificity.
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.
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
Affinity chromatography
Affinity chromatography is a type of liquid chromatography that uses reversible biological interactions or molecular recognition to separate sample components. It works by attaching a ligand with affinity for the target molecule to a stationary phase. When a sample mixture passes through the column, only molecules that bind to the ligand will be retained on the stationary phase while non-binding molecules pass through. Retained molecules can then be eluted by altering conditions to break the binding interaction selectively. Affinity chromatography is useful for purifying and studying enzymes, proteins, and other biomolecules due to its high specificity and ability to achieve a high degree of purity.
Affinity chromatography AND tissue plasminogen activator
Affinity chromatography is used to purify tissue plasminogen activator (tPA) by exploiting its specific binding to ligands. tPA breaks down blood clots and is used to treat strokes. It can be produced recombinantly in mammalian cells like CHO cells or bacteria. The ligand is immobilized on a chromatographic medium like agarose beads. tPA binds selectively to the ligand and is later eluted by altering buffer conditions like pH or ionic strength. Affinity chromatography allows for the efficient purification of tPA.
Polymeric nanoparticles A Novel Approach
This document provides an overview of polymeric nanoparticles (PNPs). It defines PNPs and explains that drugs can be dissolved, entrapped, encapsulated, or attached to the nanoparticles. The advantages of PNPs for drug delivery are described, such as increased drug stability and targeting. Methods for preparing PNPs are outlined, including polymerization, precipitation, and cross-linking techniques. Characterization methods and applications of PNPs are also summarized briefly.
Affinity chromatography
Affinity chromatography is a method used to purify biomolecules like proteins and nucleic acids based on specific interactions between the biomolecule and a ligand immobilized on a solid support. When a mixture is passed through the column, the target biomolecule will bind to the ligand while other molecules pass through. The bound biomolecule can then be separated by changing conditions like pH or introducing a competing molecule to displace it. Affinity chromatography offers highly specific purification of target molecules in a single step.
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Analytical techniques for separation or purification of proteins
Analytical techniques for separation or purification of proteins
Purification of proteins (purification of enzymes)
Purification of proteins (purification of enzymes)
Affinity chromatography AND tissue plasminogen activator
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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.
Proteins purification
This document discusses various methods for purifying proteins, including selecting an appropriate source, lysing cells, centrifugation, chromatography, electrophoresis, and measuring protein activity. Key steps involve disrupting cells to release proteins, separating components by techniques that exploit differences in properties like size, charge, and affinity for ligands, and ensuring the purified protein retains its native structure and activity. The goal is to enrich the target protein away from other biomolecules to obtain a pure sample for downstream applications.
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- 1. NADIR ULLAH DEPARTMENT OF MICROBIOLOGY BS 6TH SUBMITTED TO: DR BAHAR ULLAH
- 2. TOPIC: PURIFICATION OF ENZYME
- 3. OUTLINE • Introduction • Removal of nucleic acid and debris • Preliminary purification • Separation based on solubility • Perception • Adsorption • Final purification
- 4. INTRODUCTION • Enzyme purification is complex process and various step are to be conducted in sequence for the purification of any enzyme • Enzyme purification such method should be used which can give high degree of purity • High overall recovery and reproducibility • Nucleic acid cell debris preliminary purification and concentration steps should be followed first then final purification is done, finally concentrated and purified enzyme is obtained
- 5. REMOVAL OF NUCLEIC ACID • The release of nucleic acid makes the solution more viscous because it contains broken cell parts as major contaminant • The removal of nucleic acid and cell debris is done either by differential sedimentation or perception, and can be affected by centrifugation or filtration • Protamine sulphate streptomycin are used as precipitants
- 6. REMOVAL OF NUCLEIC ACID II • Digestion with nuclease may reduce the viscosity of the solution and can be removed in later stages of purification • At second step ,the supernatant which contains the enzyme be further subjected to process for the removal of small organic and inorganic molecule, other protein, unwanted contaminant and water
- 7. PRELIMINARY PURIFICATION • Success of purification depends on picking a number of procedures and combing them in an effective manner. • Procedure is combined to purify the specific enzymes from crude whole cell extracts.
- 8. SEPARATION BASED ON SOLUBILITY • Solubility based separation is mainly based on precipitation • A crude extract of protein is made the mixtures of different fractions are separated, involving precipitation steps • The solvent should be selected in such a way that it should not after the solubility and the structure of protein • It is also necessary to control pH, temperature and the composition of solvent
- 9. SEPARATION BASED ON SOLUBILITY II • At isoelectronic point proteins show a minimum solubility. • The decrease in solubility at the isoelectronic point shows that the individual proteins molecules which all have similar charges at pH values away from isoelectronic point) cease to repel.
- 10. PRECIPITATION • The solubility of proteins depends on the concentration of salts in the medium. • The solvent properties are changed by specific interaction between charged side chain and the solution ions, or particularly at high salt concentration. • Salt together with the possibility of slow loss ammonia , effects the change in the pH. • Sodium sulphate is an effective salt, it has low solubility phosphates and used as buffers in the biological pH range.
- 11. ADSORPTION • Crude extract processing is performed by batch adsorption and elution in initial stages of purification and concentration. • Resins, substituted dextran's or agarose, and substituted cellulose are used as adsorbents on small scale. • Hydroxyapatite is also used because of its low cost. • Differential elution of many bound proteins can be performed with increasing concentration of phosphate.
- 12. ADSORPTION II • A common feature of all adsorption techniques is the principle of adsorption, followed by adsorption or elution. • Separation is achieved by adsorption or elution of either enzymes or impurities.
- 13. FINAL PURIFICATION • Chromatography technique is the most widely used for the isolation of enzyme. • Ultrafiltration method is also used for separation of an abundant phosphate and PEG from enzyme. • It lowers the ionic strength for the following batch wise adsorption to an ion exchanger. • After desorption by decreasing salt gradient the enzyme solution may be too diluted and must be concentrated before following get filtration.
- 14. FINAL PURIFICATION II • Concentration may again be done by ultrafiltration or by precipitation with ammonium sulphate or an organic solvent, which permits further fractionation. • Dye or affinity chromatography for may also be applied. • For highest purity preparations electro focusing may be followed as the final step.
- 15. REFRENCES • Nelson, DL; Cox, MM (2009). Lehninger: Principios de bio química (5th ed.). Barcelona: Omega. pp. 220– 228. ISBN 978-84-282-1486-5. • Jump up Copley, SD (July 2012). "Moonlighting is Mainstream: Paradigm Adjustment Required". Bio Essays 34 (7): 578–588.doi:10.1002/bies.201100191