58 Questions
What does specific activity measure in an enzyme?
The quantity of active enzyme present
How is enzyme activity defined?
Moles of substrate converted per unit time
What is the purpose of differential centrifugation in protein extraction?
To recover a clean protein fraction
Which technique is commonly used to 'salt out' proteins during purification?
Ammonium sulfate
Why is it important to purify a protein?
To characterize its function, activity, and structure
What is the basis of column chromatography separation?
Overall charge
Which technique is commonly used in protein purification to separate proteins based on physical and chemical properties?
Ion-exchange chromatography
How does gel-filtration chromatography separate molecules?
Based on molecular weight
What is the primary goal in protein purification?
To keep as much of the target protein as possible
What is the primary purpose of mass spectrometry in proteomics?
Identify proteins based on mass-to-charge ratio
Which method is commonly used for determining the primary structure of proteins?
Edman Method
What technique is used to track protein activity after each purification step?
Enzyme assay
What is the purpose of raising antibodies in protein purification?
For use in assays
What is the purpose of N-terminal sequencing in protein identification?
To identify the target protein sequence
Which characteristic is NOT typically considered when determining protein purity?
Total protein concentration
What does size-exclusion chromatography primarily separate based on?
Molecular weight
In which technique are proteins separated based on their size and charge?
Gel electrophoresis
What is a common application of mass spectrometry in proteomics?
Detecting post-translational modifications
Which chromatography technique involves binding interactions for separation?
Affinity chromatography
What is the significance of isoelectric focussing in protein analysis?
Separating proteins based on charge differences
Which type of chromatography uses a gel material that doesn't interact with proteins?
Size-Exclusion Chromatography
What is the main factor that determines the pore size in Size-Exclusion Chromatography?
Extent of cross-linking
In Ion Exchange Chromatography, what type of molecules bind to negatively charged groups in the column resin?
Positively charged proteins
What happens to proteins with no net charge or a net negative charge in Ion Exchange Chromatography?
They pass through the column
How are proteins eluted in Ion Exchange Chromatography after binding to the column resin?
By adding excess Na+ ions
Which chromatography technique is based on interaction with overall charge and is less specific than affinity chromatography?
Ion Exchange Chromatography
What is used as an eluent in cation exchange chromatography?
+vely charged groups
Which type of exchange chromatography binds proteins based on negatively charged groups?
-vely charged proteins
What is the role of Na+ ions in Ion Exchange Chromatography after proteins bind to the column resin?
Displace the bound proteins from the resin
What is a characteristic of the gel material used in Size-Exclusion Chromatography?
It has uniform pores throughout the column
What does specific activity measure in an enzyme?
Amount of enzyme per milligram of total protein
What is the purpose of differential centrifugation in protein extraction?
Separate unbroken cells from cell lysate
In column chromatography, which phase interacts with the samples to be separated?
Stationary phase
What is the primary goal of gel-filtration chromatography?
Separate molecules based on size
What is a common application of mass spectrometry in proteomics?
Identification of target proteins
Which technique is commonly used to track protein activity after each purification step?
Western blot
What happens in salting out during protein purification?
Proteins precipitate due to reduced solubility
What does enzyme activity measure?
Moles of substrate converted per unit time
What is the basis of size-exclusion chromatography separation?
Molecule size differences
What is the role of N-terminal sequencing in protein identification?
Determine protein structure sequence
What is the primary reason for characterizing a protein?
To determine its function, activity, and structure
Which technique involves separating proteins based on their isoelectric points?
Gel electrophoresis
What is the main objective of protein purification according to the provided guidelines?
Minimize the number of purification steps
In protein purification, what is the purpose of developing analytical assays?
To monitor and assess the purification process
Which factor determines how pure a protein needs to be for therapeutic use in vivo studies?
> 99% purity
What method is commonly used for determining the primary structure of proteins?
N-terminal sequencing
Which chromatography technique involves separating proteins based on their binding interactions?
Affinity chromatography
What is a common application of mass spectrometry in proteomics?
Identification of proteins in a sample
What is the primary purpose of using a gel material in Size-Exclusion Chromatography?
To control the pore size for separating proteins by size
Which type of chromatography is less specific than affinity chromatography and relies on interactions based on overall charge?
Ion Exchange Chromatography
What happens to proteins that have a net positive charge in Ion Exchange Chromatography?
They bind to negatively charged groups in the column resin
In Gel Filtration Chromatography, what role does the gel play in the separation process?
Controls pore size for molecule separation
What is the primary function of raising antibodies during protein purification?
To bind specific proteins for isolation
Which technique is typically used to separate proteins based on physical and chemical properties during purification?
Column Chromatography
What is the main factor that determines the elution time of smaller molecules in Size-Exclusion Chromatography?
Pore size of the gel material
In Ion Exchange Chromatography, what happens when an excess of Na+ ions is added to the column?
Na+ ions compete with proteins for binding sites on the resin.
What characteristic of agarose, polyacrylamide, starch, or dextran makes them suitable for use in Size-Exclusion Chromatography?
Lack of interaction with proteins
What is a common application of mass spectrometry in proteomics?
Identifying post-translational modifications
Study Notes
Enzyme Activity
- Specific activity measures the number of moles of substrate converted per unit time per unit of enzyme, usually expressed as micromoles per minute per milligram of enzyme.
- Enzyme activity is defined as the rate of enzymatic conversion of substrate into product.
Protein Extraction
- Differential centrifugation is used to separate cell components based on their density and size, resulting in the separation of proteins from other cellular components.
Protein Purification
- Purification is necessary to remove contaminants, improve protein stability, and understand protein function.
- The primary goal of protein purification is to isolate a protein from its native source or a recombinant source.
Chromatography
- Column chromatography separates proteins based on their physical and chemical properties, such as size, charge, and binding affinity.
- Gel-filtration chromatography separates molecules based on their size, with smaller molecules eluting later than larger molecules.
- Size-exclusion chromatography separates based on the size of the molecule, with larger molecules being excluded from the gel matrix and eluting first.
- Ion Exchange Chromatography separates proteins based on their charge, with proteins binding to the column resin and eluting with a change in salt concentration or pH.
Protein Identification
- N-terminal sequencing is used to determine the primary structure of proteins.
- Mass spectrometry is used to determine the primary structure of proteins and identify protein modifications.
Protein Analysis
- Isoelectric focusing separates proteins based on their isoelectric points, which is the pH at which the protein has no net charge.
- Cation exchange chromatography binds proteins based on negatively charged groups, and anions are used as an eluent to elute bound proteins.
Protein Purification Techniques
- Salting out involves adding high concentrations of salt to a protein solution, causing proteins to precipitate out of solution.
- Affinity chromatography separates proteins based on specific binding interactions, such as antibody-antigen interactions.
- Gel material used in Size-Exclusion Chromatography is porous, allowing smaller molecules to enter the pores and be separated from larger molecules.
General
- The primary reason for characterizing a protein is to understand its function and biological role.
- Developing analytical assays is necessary to track protein activity during purification and to validate protein purity.
Learn about the differences between activity and specific activity in enzyme analysis. Understand which number should go up and which should go down when comparing the two metrics. Explore how enzyme activity and specific activity are calculated and their significance in biochemical studies.
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