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Questions and Answers
What is the primary purpose of generating a crude extract in protein purification?
What is the primary purpose of generating a crude extract in protein purification?
To obtain the protein of interest from the source material while preserving its structure.
Name two factors that can affect the precipitation of proteins in bioprocessing.
Name two factors that can affect the precipitation of proteins in bioprocessing.
pH and temperature.
How is the molecular weight of a protein determined?
How is the molecular weight of a protein determined?
By summing the masses of the individual amino acids composing the protein.
What determines the basic function of a protein?
What determines the basic function of a protein?
What distinguishes precipitates from crystals in protein purification?
What distinguishes precipitates from crystals in protein purification?
What role do buffers and inhibitors play during the extraction of proteins?
What role do buffers and inhibitors play during the extraction of proteins?
Define quaternary structure in proteins.
Define quaternary structure in proteins.
Provide an example of a protein with quaternary structure and describe its composition.
Provide an example of a protein with quaternary structure and describe its composition.
Can you list two commonly used methods for protein purification?
Can you list two commonly used methods for protein purification?
What is meant by the term 'salting out' in protein purification?
What is meant by the term 'salting out' in protein purification?
What are conformational changes in proteins?
What are conformational changes in proteins?
Why is it important to perform protein purification at controlled conditions?
Why is it important to perform protein purification at controlled conditions?
Why is sample preparation important in protein analysis?
Why is sample preparation important in protein analysis?
What is the first step in the purification of proteins?
What is the first step in the purification of proteins?
How do thermal vibrations affect protein structure?
How do thermal vibrations affect protein structure?
What role do assays play in protein purification?
What role do assays play in protein purification?
What defines the secondary structure of a protein?
What defines the secondary structure of a protein?
How does an alpha helix structure form in proteins?
How does an alpha helix structure form in proteins?
What stabilizes beta pleated sheet structures in proteins?
What stabilizes beta pleated sheet structures in proteins?
What is meant by the term tertiary structure in proteins?
What is meant by the term tertiary structure in proteins?
Describe the role of hydrophobic interactions in protein folding.
Describe the role of hydrophobic interactions in protein folding.
How do hydrogen bonds contribute to tertiary structure stabilization?
How do hydrogen bonds contribute to tertiary structure stabilization?
What is a disulfide bridge and its significance in protein structure?
What is a disulfide bridge and its significance in protein structure?
Explain how van der Waals forces contribute to protein stability.
Explain how van der Waals forces contribute to protein stability.
Why is the solubility of a protein lowest at its isoelectric point?
Why is the solubility of a protein lowest at its isoelectric point?
What is the effect of low salt concentrations on protein solubility?
What is the effect of low salt concentrations on protein solubility?
How do divalent ions compare to monovalent ions in terms of protein precipitation?
How do divalent ions compare to monovalent ions in terms of protein precipitation?
What role does ammonium sulfate play in protein precipitation?
What role does ammonium sulfate play in protein precipitation?
What happens during the process of centrifugation?
What happens during the process of centrifugation?
What is the purpose of differential centrifugation?
What is the purpose of differential centrifugation?
Describe the dialysis procedure for protein solutions.
Describe the dialysis procedure for protein solutions.
What is the primary reason for protein solubility decreasing at high salt concentrations?
What is the primary reason for protein solubility decreasing at high salt concentrations?
What are the primary factors that influence the migration rate of biological molecules during electrophoresis?
What are the primary factors that influence the migration rate of biological molecules during electrophoresis?
Explain the purpose of using a gel matrix in electrophoresis.
Explain the purpose of using a gel matrix in electrophoresis.
What technique can be used to visualize proteins after electrophoresis?
What technique can be used to visualize proteins after electrophoresis?
Describe the main principle behind paper electrophoresis.
Describe the main principle behind paper electrophoresis.
What role does the electric field play in gel electrophoresis?
What role does the electric field play in gel electrophoresis?
How do larger molecules behave compared to smaller molecules in a polyacrylamide gel?
How do larger molecules behave compared to smaller molecules in a polyacrylamide gel?
What is the significance of using nitrocellulose membranes in conjunction with gels during protein analysis?
What is the significance of using nitrocellulose membranes in conjunction with gels during protein analysis?
What is the term used to describe the distinct bands formed on the gel during electrophoresis?
What is the term used to describe the distinct bands formed on the gel during electrophoresis?
What is the primary purpose of using different concentrations of acrylamide in gel electrophoresis?
What is the primary purpose of using different concentrations of acrylamide in gel electrophoresis?
Why is agarose preferred for separating larger nucleic acids?
Why is agarose preferred for separating larger nucleic acids?
Explain how electromotive force (EMF) influences the movement of molecules during electrophoresis.
Explain how electromotive force (EMF) influences the movement of molecules during electrophoresis.
What is the significance of maintaining a high pH (~9) in the buffer during electrophoresis of proteins?
What is the significance of maintaining a high pH (~9) in the buffer during electrophoresis of proteins?
In chromatography, what role does the stationary phase play in the separation of molecules?
In chromatography, what role does the stationary phase play in the separation of molecules?
What factors influence the separation of molecules in chromatography?
What factors influence the separation of molecules in chromatography?
Describe the composition of the stationary phase in chromatography.
Describe the composition of the stationary phase in chromatography.
How does the size of a molecule affect its electrophoretic mobility in a gel?
How does the size of a molecule affect its electrophoretic mobility in a gel?
Flashcards
Tertiary Structure
Tertiary Structure
The three-dimensional structure of a single polypeptide chain, formed by interactions between its amino acid side chains.
Quaternary Structure
Quaternary Structure
The arrangement of multiple polypeptide chains (subunits) in a protein complex.
Homodimer
Homodimer
A protein complex that consists of multiple identical subunits.
Heterodimer
Heterodimer
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Conformational Change
Conformational Change
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Protein Purification
Protein Purification
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Protein Assay
Protein Assay
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Extraction
Extraction
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Protein Secondary Structure
Protein Secondary Structure
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Alpha (α) helix
Alpha (α) helix
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Beta (β) pleated sheet
Beta (β) pleated sheet
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Protein Tertiary Structure
Protein Tertiary Structure
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Hydrophobic Interactions
Hydrophobic Interactions
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Hydrogen Bonding in Tertiary Structure
Hydrogen Bonding in Tertiary Structure
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Ionic Bonding in Tertiary Structure
Ionic Bonding in Tertiary Structure
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Disulfide Bridges
Disulfide Bridges
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Isoelectric Point
Isoelectric Point
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Salting-In
Salting-In
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Salting-Out
Salting-Out
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Centrifugation
Centrifugation
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Pellet
Pellet
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Supernatant
Supernatant
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Differential Centrifugation
Differential Centrifugation
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Dialysis
Dialysis
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Obtaining Source Material
Obtaining Source Material
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What is a Crude Extract?
What is a Crude Extract?
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Why Use Buffers and Inhibitors?
Why Use Buffers and Inhibitors?
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What is Precipitation in Protein Purification?
What is Precipitation in Protein Purification?
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What is Salting Out?
What is Salting Out?
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Factors Affecting Salting Out
Factors Affecting Salting Out
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What is Crystallization?
What is Crystallization?
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What is Affinity Chromatography?
What is Affinity Chromatography?
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Electrophoresis
Electrophoresis
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Gel Matrix
Gel Matrix
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Electrophoretic Mobility
Electrophoretic Mobility
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Molecular Sieve Effect
Molecular Sieve Effect
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Protein Staining
Protein Staining
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Western Blot
Western Blot
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PAGE (Polyacrylamide Gel Electrophoresis)
PAGE (Polyacrylamide Gel Electrophoresis)
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Native Gel
Native Gel
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What is electrophoresis?
What is electrophoresis?
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What is an electrophoresis gel?
What is an electrophoresis gel?
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What is SDS-PAGE?
What is SDS-PAGE?
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What is the stationary phase in chromatography?
What is the stationary phase in chromatography?
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What is the mobile phase in chromatography?
What is the mobile phase in chromatography?
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How does chromatography separate molecules?
How does chromatography separate molecules?
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What factors influence separation in chromatography?
What factors influence separation in chromatography?
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What are the key components of chromatography?
What are the key components of chromatography?
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Study Notes
Proteins
- Proteins are macromolecules formed by amino acids.
- There are 20 different amino acids in proteins.
- Amino acids are linked together by peptide linkages to form polypeptide chains.
- A protein can contain one or more long polypeptide chains.
- Short chains of amino acids (fewer than 20-30 residues) are called peptides or oligopeptides.
- The sequence of amino acids in a protein is defined by the genetic code.
- Amino acid residues in proteins can be chemically modified after synthesis (post-translational modification).
- Proteins may have non-peptide prosthetic groups or cofactors.
Protein Structure
- Proteins have four levels of structure: primary, secondary, tertiary, and quaternary.
- Primary structure is the unique order of amino acids in a protein.
- Secondary structure involves regularly repeating local structures stabilized by hydrogen bonds. This includes alpha helices and beta pleated sheets.
- Tertiary structure is the overall 3D shape of a single protein molecule, formed by interactions between secondary structures. It includes hydrogen bonds, disulfide bridges, hydrophobic interactions, and ionic bonds.
- Quaternary structure refers to the structure of a protein formed by interactions between multiple polypeptide chains. These are called subunits.
Protein Methods of Isolation, Purification, and Identification
- Sample Preparation: Treating samples to prevent contamination and improve accuracy before analysis. This often starts with extraction.
- Assays: Used to identify the protein of interest after fractionation. These can be spectroscopic (Bradford reagent, chromagenic substrate) or immunological (antibodies).
- Crude Extracts: Obtaining the material containing the protein of interest, then preparing a crude extract. This might involve grinding, breaking open cells, etc. This is done in a buffer with inhibitors.
- Protein Purification Methods: Using differences in physico-chemical properties (molecular size, charge, solubility, etc.) to purify proteins from the crude extract. Techniques include precipitation, centrifugation, chromatography (gel filtration, ion exchange, affinity), electrophoresis, and crystallization.
Precipitation
- Precipitation is a method for purifying or concentrating proteins.
- It involves using solvents, salts, or increased temperature to precipitate proteins out of solution.
- "Salting out" is the use of salts (like ammonium sulfate) to reduce the solubility of proteins and cause them to precipitate.
- Effective precipitation methods are dependent on many factors, including pH, temperature, protein concentration, and the salt used or other precipitation agent.
Centrifugation
- Centrifugation is used to separate mixtures based on density differences.
- It forces the denser components of the mixture to the bottom of the tube (precipitate).
- Differential centrifugation involves multiple rounds of centrifugation at increasing speeds to separate different cellular or macromolecular components.
Dialysis
- Dialysis is a technique used to exchange the solvent surrounding a protein.
- It uses a semi-permeable membrane that allows water and small molecules to pass through but prevents the protein from leaving.
- The purpose of dialysis is to change the composition of the solution around the protein (e.g. a buffer change).
Chromatography
- Chromatography is a powerful fractionation method used to separate components of a mixture.
- In Chromatography a mixture is applied to a stationary phase, and a mobile phase moves over the stationary phase.
- The separation is based on differences in affinity to the stationary phase. Types include Gel Filtration, Ion Exchange, and Affinity.
- Gel Filtration/Size Exclusion: Separates molecules based on size. Larger molecules exit the column first.
- Ion Exchange: Separates molecules based on charge.
- Affinity: Separates molecules based on their specific binding affinity for a ligand (e.g., antibody, enzyme).
Electrophoresis
- Electrophoresis separates molecules based on their size and charge as they move through a gel or other matrix in an electric field.
- It's used for identification, sequencing, or further manipulation.
- PAGE (PolyAcrylamide Gel Electrophoresis): Separation of proteins based on their size and charge.
Chromatography
- Different Chromatography techniques are used for various classes of compounds.
- Paper Chromatography: A simple method for separating small molecules based on their different properties.
- Column Chromatography: Used to separate components of a mixture based on their different affinities for the stationary phase.
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Description
Explore the fascinating world of proteins, their formation, and structural levels. This quiz covers essential concepts such as amino acids, peptide linkages, and protein structure including primary, secondary, tertiary, and quaternary forms. Test your knowledge and understanding of these vital macromolecules!