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Questions and Answers
What is the primary principle behind differential centrifugation?
What is the primary principle behind differential centrifugation?
In rate zonal centrifugation, how are substances ideally separated?
In rate zonal centrifugation, how are substances ideally separated?
What does a uniform band in density gradient centrifugation indicate?
What does a uniform band in density gradient centrifugation indicate?
What mechanism does dialysis primarily use for separation?
What mechanism does dialysis primarily use for separation?
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How does diffusion play a role in dialysis?
How does diffusion play a role in dialysis?
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What would be a likely outcome if centrifugation is performed at a higher speed?
What would be a likely outcome if centrifugation is performed at a higher speed?
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Which of the following statements best describes the role of a selectively permeable membrane in dialysis?
Which of the following statements best describes the role of a selectively permeable membrane in dialysis?
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Which centrifugation technique would most likely achieve optimal results for separating similar density substances?
Which centrifugation technique would most likely achieve optimal results for separating similar density substances?
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What is the primary purpose of Edman degradation?
What is the primary purpose of Edman degradation?
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In which step of mass spectrometry does ionized peptides get accelerated?
In which step of mass spectrometry does ionized peptides get accelerated?
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Which reagent is commonly used in chemical synthesis of peptides?
Which reagent is commonly used in chemical synthesis of peptides?
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What does the first step of Western blotting involve?
What does the first step of Western blotting involve?
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What is the result of complete hydrolysis of peptide bonds during protein analysis?
What is the result of complete hydrolysis of peptide bonds during protein analysis?
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What is the primary function of affinity chromatography?
What is the primary function of affinity chromatography?
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What could enhance the elution of non-binding proteins in affinity chromatography?
What could enhance the elution of non-binding proteins in affinity chromatography?
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What is the role of a competitive inhibitor in affinity chromatography?
What is the role of a competitive inhibitor in affinity chromatography?
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What is the primary principle behind size exclusion chromatography?
What is the primary principle behind size exclusion chromatography?
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How does exchange chromatography separate molecules?
How does exchange chromatography separate molecules?
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In size exclusion chromatography, how do larger molecules behave in the separation process?
In size exclusion chromatography, how do larger molecules behave in the separation process?
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What is the role of beads in size exclusion chromatography?
What is the role of beads in size exclusion chromatography?
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What influences the strength of interaction in exchange chromatography?
What influences the strength of interaction in exchange chromatography?
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Under what pH conditions would a protein have a negative charge in exchange chromatography?
Under what pH conditions would a protein have a negative charge in exchange chromatography?
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What determines the resolution of separation in size exclusion chromatography?
What determines the resolution of separation in size exclusion chromatography?
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What happens to the protein structure during the elution process in affinity chromatography?
What happens to the protein structure during the elution process in affinity chromatography?
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Which technique could effectively analyze protein purity?
Which technique could effectively analyze protein purity?
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What type of buffer is added to ensure proteins are protonated during affinity chromatography?
What type of buffer is added to ensure proteins are protonated during affinity chromatography?
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When smaller molecules are trapped inside the beads during chromatography, what effect does this have on their elution time?
When smaller molecules are trapped inside the beads during chromatography, what effect does this have on their elution time?
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In the context of chromatography, what implies 'stronger interaction'?
In the context of chromatography, what implies 'stronger interaction'?
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In the context of Fast Protein Liquid Chromatography (FPLC), what does a peak represent?
In the context of Fast Protein Liquid Chromatography (FPLC), what does a peak represent?
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What might be a consequence of using inappropriate bead size in size exclusion chromatography?
What might be a consequence of using inappropriate bead size in size exclusion chromatography?
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What characterizes the elution order of proteins in exchange chromatography?
What characterizes the elution order of proteins in exchange chromatography?
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Which statement accurately describes the behavior of smaller molecules during the chromatography process?
Which statement accurately describes the behavior of smaller molecules during the chromatography process?
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Which of the following best describes the utility of different columns in size exclusion chromatography?
Which of the following best describes the utility of different columns in size exclusion chromatography?
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What is the primary purpose of SDS in the SDS-PAGE process?
What is the primary purpose of SDS in the SDS-PAGE process?
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In non-denaturing gel electrophoresis, proteins are separated based on which of the following?
In non-denaturing gel electrophoresis, proteins are separated based on which of the following?
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How does the migration of proteins during isoelectric focusing occur?
How does the migration of proteins during isoelectric focusing occur?
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Which staining method is commonly used for visualizing proteins after electrophoresis?
Which staining method is commonly used for visualizing proteins after electrophoresis?
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What type of proteins can be separated using Western blotting combined with gel electrophoresis?
What type of proteins can be separated using Western blotting combined with gel electrophoresis?
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What is the significance of using mercaptoethanol in protein sample preparation for SDS-PAGE?
What is the significance of using mercaptoethanol in protein sample preparation for SDS-PAGE?
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What role does the zwitterionic form of proteins play during isoelectric focusing?
What role does the zwitterionic form of proteins play during isoelectric focusing?
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Which of the following statements about PAGE is correct?
Which of the following statements about PAGE is correct?
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What is the primary characteristic of the two-dimensional gel electrophoresis technique?
What is the primary characteristic of the two-dimensional gel electrophoresis technique?
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Study Notes
Protein Purification Techniques
-
Centrifugation: Separates substances based on density differences using centrifugal force.
- Differential centrifugation: Separates components with varying densities based on their sedimentation rates during centrifugation.
- Rate-zonal centrifugation: Separates components based on their sedimentation rates through a density gradient.
- Density gradient centrifugation: Uses a gradient of density to separate substances based on their density.
- Dialysis: Separates molecules based on size through a selectively permeable membrane. Molecules diffuse down their concentration gradient.
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Affinity Chromatography: Separates proteins based on their specific binding to a specific antibody or ligand.
- Specific protein binds to an antibody or similar component in the first eluent, and non-binding proteins elute out.
- pH or salt concentration can change the protein protonation or break bonds, releasing the protein of interest.
- Competitive inhibitors or changing buffer strength can help separate the desired molecule from others.
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Exchange Chromatography: Separates molecules based on their electrical charges.
- The strength of interaction between the protein and an ion exchange matrix depends on the charge density on the protein.
- pH above PI yields a negative charge for the protein, and pH below PI yields a positive charge.
Protein Detection & Characterization
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Polyacrylamide Gel Electrophoresis (PAGE): Separates proteins based on size.
- SDS-PAGE (Denaturing): Uses SDS to create a uniform negative charge on proteins, so separation occurs primarily by size.
- Non-denaturing: Separates based on charge and size/conformation.
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Western Blotting: Method for detecting specific proteins within a mixture of proteins using antibodies.
- Uses antibodies to detect and identify proteins of interest in a mixture.
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Edman Degradation: Method for determining the amino acid sequence of small peptides.
- Enables the sequencing of peptides to establish protein sequence.
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Mass Spectrometry: Determines the amino acid sequence.
- Ionization, acceleration, and deflection stages lead to detection.
- Chemical Synthesis of Peptides: Used to create short chains of amino acids (peptides) used in research.
- X-Ray Crystallography: Determines 3D structure of proteins.
- NMR Spectroscopy: Analyzes small to medium-sized proteins to understand their structure.
- Cryo-EM (Cryo-electron Microscopy): Used to determine the structure of larger proteins and complexes. Proteins are frozen to help stabilize their structure in vacuum.
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Description
Explore the various techniques used in protein purification, including centrifugation, dialysis, and affinity chromatography. This quiz covers methods such as differential and density gradient centrifugation, and their applications in separating proteins based on size and binding properties.
