Enzyme Activity vs Specific Activity Quiz and Flashcards

Questions and Answers

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 Signup and view all the answers

Why is it important to purify a protein?

To characterize its function, activity, and structure Signup and view all the answers

What is the basis of column chromatography separation?

Overall charge Signup and view all the answers

Which technique is commonly used in protein purification to separate proteins based on physical and chemical properties?

Ion-exchange chromatography Signup and view all the answers

How does gel-filtration chromatography separate molecules?

Based on molecular weight Signup and view all the answers

What is the primary goal in protein purification?

To keep as much of the target protein as possible Signup and view all the answers

What is the primary purpose of mass spectrometry in proteomics?

Identify proteins based on mass-to-charge ratio Signup and view all the answers

Which method is commonly used for determining the primary structure of proteins?

Edman Method Signup and view all the answers

What technique is used to track protein activity after each purification step?

Enzyme assay Signup and view all the answers

What is the purpose of raising antibodies in protein purification?

For use in assays Signup and view all the answers

What is the purpose of N-terminal sequencing in protein identification?

To identify the target protein sequence Signup and view all the answers

Which characteristic is NOT typically considered when determining protein purity?

Total protein concentration Signup and view all the answers

What does size-exclusion chromatography primarily separate based on?

Molecular weight Signup and view all the answers

In which technique are proteins separated based on their size and charge?

Gel electrophoresis Signup and view all the answers

What is a common application of mass spectrometry in proteomics?

Detecting post-translational modifications Signup and view all the answers

Which chromatography technique involves binding interactions for separation?

Affinity chromatography Signup and view all the answers

What is the significance of isoelectric focussing in protein analysis?

Separating proteins based on charge differences Signup and view all the answers

Which type of chromatography uses a gel material that doesn't interact with proteins?

Size-Exclusion Chromatography Signup and view all the answers

What is the main factor that determines the pore size in Size-Exclusion Chromatography?

Extent of cross-linking Signup and view all the answers

In Ion Exchange Chromatography, what type of molecules bind to negatively charged groups in the column resin?

Positively charged proteins Signup and view all the answers

What happens to proteins with no net charge or a net negative charge in Ion Exchange Chromatography?

They pass through the column Signup and view all the answers

How are proteins eluted in Ion Exchange Chromatography after binding to the column resin?

By adding excess Na+ ions Signup and view all the answers

Which chromatography technique is based on interaction with overall charge and is less specific than affinity chromatography?

Ion Exchange Chromatography Signup and view all the answers

What is used as an eluent in cation exchange chromatography?

+vely charged groups Signup and view all the answers

Which type of exchange chromatography binds proteins based on negatively charged groups?

-vely charged proteins Signup and view all the answers

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 Signup and view all the answers

What is a characteristic of the gel material used in Size-Exclusion Chromatography?

It has uniform pores throughout the column Signup and view all the answers

What does specific activity measure in an enzyme?

Amount of enzyme per milligram of total protein Signup and view all the answers

What is the purpose of differential centrifugation in protein extraction?

Separate unbroken cells from cell lysate Signup and view all the answers

In column chromatography, which phase interacts with the samples to be separated?

Stationary phase Signup and view all the answers

What is the primary goal of gel-filtration chromatography?

Separate molecules based on size Signup and view all the answers

What is a common application of mass spectrometry in proteomics?

Identification of target proteins Signup and view all the answers

Which technique is commonly used to track protein activity after each purification step?

Western blot Signup and view all the answers

What happens in salting out during protein purification?

Proteins precipitate due to reduced solubility Signup and view all the answers

What does enzyme activity measure?

Moles of substrate converted per unit time Signup and view all the answers

What is the basis of size-exclusion chromatography separation?

Molecule size differences Signup and view all the answers

What is the role of N-terminal sequencing in protein identification?

Determine protein structure sequence Signup and view all the answers

What is the primary reason for characterizing a protein?

To determine its function, activity, and structure Signup and view all the answers

Which technique involves separating proteins based on their isoelectric points?

Gel electrophoresis Signup and view all the answers

What is the main objective of protein purification according to the provided guidelines?

Minimize the number of purification steps Signup and view all the answers

In protein purification, what is the purpose of developing analytical assays?

To monitor and assess the purification process Signup and view all the answers

Which factor determines how pure a protein needs to be for therapeutic use in vivo studies?

> 99% purity Signup and view all the answers

What method is commonly used for determining the primary structure of proteins?

N-terminal sequencing Signup and view all the answers

Which chromatography technique involves separating proteins based on their binding interactions?

Affinity chromatography Signup and view all the answers

What is a common application of mass spectrometry in proteomics?

Identification of proteins in a sample Signup and view all the answers

What is the primary purpose of using a gel material in Size-Exclusion Chromatography?

To control the pore size for separating proteins by size Signup and view all the answers

Which type of chromatography is less specific than affinity chromatography and relies on interactions based on overall charge?

Ion Exchange Chromatography Signup and view all the answers

What happens to proteins that have a net positive charge in Ion Exchange Chromatography?

They bind to negatively charged groups in the column resin Signup and view all the answers

In Gel Filtration Chromatography, what role does the gel play in the separation process?

Controls pore size for molecule separation Signup and view all the answers

What is the primary function of raising antibodies during protein purification?

To bind specific proteins for isolation Signup and view all the answers

Which technique is typically used to separate proteins based on physical and chemical properties during purification?

Column Chromatography Signup and view all the answers

What is the main factor that determines the elution time of smaller molecules in Size-Exclusion Chromatography?

Pore size of the gel material Signup and view all the answers

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. Signup and view all the answers

What characteristic of agarose, polyacrylamide, starch, or dextran makes them suitable for use in Size-Exclusion Chromatography?

Lack of interaction with proteins Signup and view all the answers

What is a common application of mass spectrometry in proteomics?

Identifying post-translational modifications Signup and view all the answers

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.

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Description

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.

Activity vs Specific Activity in Enzyme Analysis

Questions and Answers

What does specific activity measure in an enzyme?

How is enzyme activity defined?

What is the purpose of differential centrifugation in protein extraction?

Which technique is commonly used to 'salt out' proteins during purification?

Why is it important to purify a protein?

What is the basis of column chromatography separation?

Which technique is commonly used in protein purification to separate proteins based on physical and chemical properties?

How does gel-filtration chromatography separate molecules?

What is the primary goal in protein purification?

What is the primary purpose of mass spectrometry in proteomics?

Which method is commonly used for determining the primary structure of proteins?

What technique is used to track protein activity after each purification step?

What is the purpose of raising antibodies in protein purification?

What is the purpose of N-terminal sequencing in protein identification?

Which characteristic is NOT typically considered when determining protein purity?

What does size-exclusion chromatography primarily separate based on?

In which technique are proteins separated based on their size and charge?

What is a common application of mass spectrometry in proteomics?

Which chromatography technique involves binding interactions for separation?

What is the significance of isoelectric focussing in protein analysis?

Which type of chromatography uses a gel material that doesn't interact with proteins?

What is the main factor that determines the pore size in Size-Exclusion Chromatography?

In Ion Exchange Chromatography, what type of molecules bind to negatively charged groups in the column resin?

What happens to proteins with no net charge or a net negative charge in Ion Exchange Chromatography?

How are proteins eluted in Ion Exchange Chromatography after binding to the column resin?

Which chromatography technique is based on interaction with overall charge and is less specific than affinity chromatography?

What is used as an eluent in cation exchange chromatography?

Which type of exchange chromatography binds proteins based on negatively charged groups?

What is the role of Na+ ions in Ion Exchange Chromatography after proteins bind to the column resin?

What is a characteristic of the gel material used in Size-Exclusion Chromatography?

What does specific activity measure in an enzyme?

What is the purpose of differential centrifugation in protein extraction?

In column chromatography, which phase interacts with the samples to be separated?

What is the primary goal of gel-filtration chromatography?

What is a common application of mass spectrometry in proteomics?

Which technique is commonly used to track protein activity after each purification step?

What happens in salting out during protein purification?

What does enzyme activity measure?

What is the basis of size-exclusion chromatography separation?

What is the role of N-terminal sequencing in protein identification?

What is the primary reason for characterizing a protein?

Which technique involves separating proteins based on their isoelectric points?

What is the main objective of protein purification according to the provided guidelines?

In protein purification, what is the purpose of developing analytical assays?

Which factor determines how pure a protein needs to be for therapeutic use in vivo studies?

What method is commonly used for determining the primary structure of proteins?

Which chromatography technique involves separating proteins based on their binding interactions?

What is a common application of mass spectrometry in proteomics?

What is the primary purpose of using a gel material in Size-Exclusion Chromatography?

Which type of chromatography is less specific than affinity chromatography and relies on interactions based on overall charge?

What happens to proteins that have a net positive charge in Ion Exchange Chromatography?

In Gel Filtration Chromatography, what role does the gel play in the separation process?

What is the primary function of raising antibodies during protein purification?

Which technique is typically used to separate proteins based on physical and chemical properties during purification?

What is the main factor that determines the elution time of smaller molecules in Size-Exclusion Chromatography?

In Ion Exchange Chromatography, what happens when an excess of Na+ ions is added to the column?

What characteristic of agarose, polyacrylamide, starch, or dextran makes them suitable for use in Size-Exclusion Chromatography?

What is a common application of mass spectrometry in proteomics?

Study Notes

Enzyme Activity

Protein Extraction

Protein Purification

Chromatography

Protein Identification

Protein Analysis

Protein Purification Techniques

General

Studying That Suits You

Description

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