Activity vs Specific Activity in Enzyme Analysis

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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

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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