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Questions and Answers
What is the main reason for purifying a protein?
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?
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?
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?
How can charged surface residues influence protein separation during purification?
What aspect of a protein's structure can be used to assist in its purification through column chromatography?
What aspect of a protein's structure can be used to assist in its purification through column chromatography?
Why is it crucial to define the objectives before starting the protein purification process?
Why is it crucial to define the objectives before starting the protein purification process?
What technique is used to determine the primary structure of a protein?
What technique is used to determine the primary structure of a protein?
What is the goal of protein purification?
What is the goal of protein purification?
What is the specific activity of an enzyme a measure of?
What is the specific activity of an enzyme a measure of?
Which process is essential before protein purification can begin?
Which process is essential before protein purification can begin?
What is the purpose of Differential Centrifugation during protein extraction?
What is the purpose of Differential Centrifugation during protein extraction?
In Column Chromatography, proteins distribute themselves into which two phases?
In Column Chromatography, proteins distribute themselves into which two phases?
What is the basis for Size-Exclusion/Gel-Filtration Chromatography to separate molecules?
What is the basis for Size-Exclusion/Gel-Filtration Chromatography to separate molecules?
Which technique is commonly used to remove water and make proteins less soluble during purification?
Which technique is commonly used to remove water and make proteins less soluble during purification?
What does Specific Activity measure in an enzyme sample?
What does Specific Activity measure in an enzyme sample?
What is the primary goal of purification using column chromatography?
What is the primary goal of purification using column chromatography?
'Salting Out' makes proteins less soluble due to which type of interactions?
'Salting Out' makes proteins less soluble due to which type of interactions?
What is the main purpose of Differential Centrifugation during protein extraction?
What is the main purpose of Differential Centrifugation during protein extraction?
What is the purpose of controlling the extent of cross-linking in the stationary phase of column chromatography?
What is the purpose of controlling the extent of cross-linking in the stationary phase of column chromatography?
What happens to larger molecules in size-exclusion/gel-filtration chromatography?
What happens to larger molecules in size-exclusion/gel-filtration chromatography?
In ion exchange chromatography, what happens when an excess of Na+ ions is added to the column?
In ion exchange chromatography, what happens when an excess of Na+ ions is added to the column?
What is the main interaction principle in ion exchange chromatography?
What is the main interaction principle in ion exchange chromatography?
Which type of exchange occurs when proteins bind to positively charged groups in ion exchange chromatography?
Which type of exchange occurs when proteins bind to positively charged groups in ion exchange chromatography?
What is used as an eluent in cationic exchange in ion exchange chromatography?
What is used as an eluent in cationic exchange in ion exchange chromatography?
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.
