32 Questions
What is the purpose of protein purification?
To isolate a pure preparation of a protein for property determination
Which technique is NOT used in the preliminary steps of protein purification?
Gel filtration chromatography
What is an essential factor impacting the speed of elution in protein chromatography?
The size of the column packing particles
Which approach is commonly employed to break open cells during protein extraction?
Homogenization
In protein purification by ion exchange chromatography, which peptide would elute first from a cation-exchange resin?
Peptide A with a pI of 5.1
Which factor plays a crucial role in determining the elution order of peptides in ion exchange chromatography?
Charge of the peptides
How does the presence of Glu and Asp residues in a peptide affect its elution behaviour in ion exchange chromatography using cation-exchange resin?
It causes the peptide to elute earlier
What is the primary purpose of chromatography?
To analyze or separate mixtures of gases, liquids, or dissolved substances
Which type of chromatography involves separating molecules based on their size?
Size-exclusion chromatography
What was the original focus of chromatography when it was first recorded in the mid-1800s?
Separation of plant pigments
Which field uses chromatography for athlete testing, including horses?
Forensics
What is the main principle behind affinity chromatography?
Binding affinity
In affinity chromatography, how are proteins that do not bind to the ligand treated?
They flow more rapidly through the column
What is used to elute bound proteins in affinity chromatography?
High concentration of salt
What is the effect of a free ligand in affinity chromatography?
It competes with the attached ligand, releasing bound proteins
Which type of chromatography is primarily based on bead migration rather than binding affinity?
Size exclusion chromatography
What is the main mechanism by which proteins are separated in size exclusion chromatography?
Migration through beads based on size
Salting out in protein purification involves increasing solubility of proteins by adding salts.
False
Proteins vary in size, charge, and binding properties due to genetic engineering.
False
The purpose of protein purification is only to determine the properties of proteins, not their activity.
False
Gas Chromatography (GC) is not a common method of chromatography.
False
High-Performance Liquid Chromatography (HPLC) is a type of column chromatography.
True
Thin-Layer Chromatography (TLC) does not involve a stationary phase.
False
Size Exclusion Chromatography is not a separation principle used in chromatographic purification.
False
In column chromatography, the length of the column does not impact the column efficiency.
False
The elution order of peptides in ion exchange chromatography is primarily determined by their boiling points.
False
Recombinant proteins are not used for Western Blot techniques.
False
Recombinant proteins are never used in animal models to identify therapeutic targets.
False
One of the disadvantages of recombinant proteins is their lack of post-translational modifications.
True
In hydrophobic interaction chromatography (HIC), the binding is independent of the surface hydrophobicity of the protein.
False
Proteins that do not bind a ligand in protein chromatography can be purified by altering the gene to express a fusion protein for affinity chromatography.
True
The elution order of peptides in size exclusion chromatography is primarily determined by their hydrophobicity.
False
Study Notes
- Chromatography is a technique used for analyzing or separating mixtures of substances, involving two distinct phases: stationary and mobile. (Oxford Dictionary of Science, 1999)
- Originating in the mid-1800s, chromatography was first documented for separating plant pigments. (Various sources)
- Chromatography has diverse applications, including forensics, food regulation, and athlete testing. (Various sources)
- Protein chromatography is a type of chromatography used for protein purification.
- Protein purification serves the purpose of preparing a pure protein sample to determine its properties or activity.
- Preliminary steps in protein purification include protein extraction and precipitation.
- Size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography are methods used in protein purification. (Various sources)
- Chromatography techniques differ from HPLC/GC in terms of pressure, temperature, and flow rate.
- Protein extraction involves obtaining proteins from tissue or microbial cells, which necessitates breaking open the cells.
- Ion exchange chromatography separates proteins based on their charge by utilizing resins with oppositely charged groups.
- Size exclusion chromatography separates proteins based on their size.
- Affinity chromatography utilizes a specific ligand for binding the protein of interest.
- Chromatography techniques, such as size exclusion and affinity, require calibration for accurate results.
- Affinity chromatography utilizes a ligand covalently attached to the beads, and proteins bind to the beads based on their affinity for the ligand.
- Elution in affinity chromatography is achieved by using a high concentration of salt or a free ligand.
- Size exclusion and ion exchange chromatography are commonly used chromatographic methods.
Test your knowledge on various protein purification techniques such as size-exclusion chromatography, ion-exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography. Learn about differences from HPLC/GC, low pressure, low temperature, low flow rate, and column chromatography.
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