Gel Filtration Chromatography Quiz and Flashcards

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Questions and Answers

What is the main reason for purifying a protein?

To decrease the size of the protein

To increase the yield of the protein

Characterize function, activity, and structure (correct)

To change the protein's primary structure

In protein purification, what is one way to minimize the number of steps involved?

Use a different technique at each step (correct)

Not define clear objectives

Choose a single long and complicated purification method

Combine multiple proteins together

Which technique is commonly used for protein purification based on physical and chemical properties?

Electrophoresis

Mass spectrometry

Column chromatography (correct)

Proteomics

How can charged surface residues influence protein separation during purification?

They can interact with chromatographic resins Signup and view all the answers

What aspect of a protein's structure can be used to assist in its purification through column chromatography?

Size and shape Signup and view all the answers

Why is it crucial to define the objectives before starting the protein purification process?

To ensure clarity in the purification strategy Signup and view all the answers

What technique is used to determine the primary structure of a protein?

Edman Method Signup and view all the answers

What is the goal of protein purification?

To optimize removal of non-target proteins while retaining target proteins Signup and view all the answers

What is the specific activity of an enzyme a measure of?

The quantity of active enzyme per milligram of total protein Signup and view all the answers

Which process is essential before protein purification can begin?

Homogenization Signup and view all the answers

What is the purpose of Differential Centrifugation during protein extraction?

To recover clean protein fractions by separating unbroken cells and other organelles Signup and view all the answers

In Column Chromatography, proteins distribute themselves into which two phases?

Stationary and Mobile Signup and view all the answers

What is the basis for Size-Exclusion/Gel-Filtration Chromatography to separate molecules?

Molecular weight Signup and view all the answers

Which technique is commonly used to remove water and make proteins less soluble during purification?

Salting Out Signup and view all the answers

What does Specific Activity measure in an enzyme sample?

Activity with respect to total protein content present Signup and view all the answers

What is the primary goal of purification using column chromatography?

To separate different compounds based on their affinity to the solvent Signup and view all the answers

'Salting Out' makes proteins less soluble due to which type of interactions?

Hydrophobic interactions among proteins increase Signup and view all the answers

What is the main purpose of Differential Centrifugation during protein extraction?

To separate different cellular components based on their densities Signup and view all the answers

What is the purpose of controlling the extent of cross-linking in the stationary phase of column chromatography?

To determine the pore size Signup and view all the answers

What happens to larger molecules in size-exclusion/gel-filtration chromatography?

They do not enter the pores and elute before smaller molecules 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 bound proteins for binding sites on the resin Signup and view all the answers

What is the main interaction principle in ion exchange chromatography?

Overall charge of proteins Signup and view all the answers

Which type of exchange occurs when proteins bind to positively charged groups in ion exchange chromatography?

Cation exchange Signup and view all the answers

What is used as an eluent in cationic exchange in ion exchange chromatography?

Gradient of increasing NaCl concentration Signup and view all the answers

Study Notes

Stationary Phase and Size Exclusion Chromatography

Stationary phase composed of cross-linked gel particles with controlled pore size.
Smaller molecules enter the pores and are delayed in elution time, while larger molecules do not enter and elute from the column before smaller ones.

Ion Exchange Chromatography

interaction based on overall charge (less specific than affinity).
Cation exchange: binds to +vely charged groups.
Anion exchange: binds to –vely charged groups.
Cationic exchange using a gradient of increasing concentration of NaCl as eluent.

Ion Exchange Chromatography Process

Proteins are applied to the column, and those with no net charge or a net negative charge pass through.
Proteins with a net positive charge stick to the column, displacing the Na+ counterions.
An excess of Na+ ions is added to the column, outcompeting the bound proteins for the binding sites, and the proteins elute.

Enzyme Activity and Specific Activity

Enzyme activity: moles of substrate converted per unit time = rate × reaction volume.
Specific activity: enzyme activity per milligram of total protein expressed in μmol min-1mg-1.
Specific activity increases, while total protein content decreases during purification.

Tracking Proteins during Purification

Purification is a multi-step process.
Methods to track proteins include:
- Enzyme activity assays
- Western blot or ELISA (for antibody detection)
- Size determination (not as specific)
- Mass spectrometry
- N-terminal sequencing

Protein Extraction from Cells

Many different proteins exist within one cell.
Steps needed to extract the protein of interest:
- Homogenization (physical disruption of cell to release proteins; lysis of cells)
- Differential centrifugation to recover a clean protein fraction
- Salting out (e.g., using ammonium sulfate)

Column Chromatography

Basis of chromatography: different compounds distribute themselves to a varying extent between two phases.
Proteins in solution interact with the stationary phase (resin in column) and the mobile phase (solvent passing through the column).

Purification using Column Chromatography

Size-Exclusion/Gel-Filtration chromatography: separates molecules based on size.
Ion Exchange chromatography: separates molecules based on charge.
Affinity chromatography: separates molecules based on specific interactions.

Guidelines for Protein Purification

Define objectives
Define properties of target protein and critical contaminants
Minimize the number of steps
Use a different technique at each step
Develop analytical assays

Purity of Protein

Required purity depends on the application:
- Therapeutic use, in vivo studies: extremely high (> 99%)
- Biochemical assays, X-ray crystallography: high (95-99%)
- N-terminal sequencing, antigen for antibody production, NMR: moderately high (< 95%)

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Description

Learn about gel filtration chromatography, a technique that uses a stationary phase composed of cross-linked gel particles to separate molecules based on their size. Discover how the extent of cross-linking can control pore size, allowing smaller molecules to enter pores and be delayed in elution time.

Gel Filtration Chromatography

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