36 Questions
What is the primary structure of proteins?
The linear sequence of amino acids held together by peptide bonds
What is the isoelectric point (pI) of an amino acid?
The pH at which the amino acid carries no net electrical charge
What are peptides?
Linear sequences of amino acids linked by peptide bonds
How many common amino acids are there?
20
What are proteins' various biological functions?
Serving as structural components, enzymes, immunoglobulins, and hormones
What is the zwitterion form of an amino acid?
A form with separate positively and negatively charged functional groups
What did Dutch chemist GJ Mulder observe while investigating substances in milk and eggs?
They seemed to have the same empirical formula (C20H31N5O12).
Who suggested that substances in milk and eggs should be called proteins?
Swedish chemist JJ Berzelius
What is the topic of Week 9 in the course schedule?
Protein analysis
Who are the authors of the book 'Bioanalytical Chemistry' mentioned in the text?
Andreas Manz, Petra Dittrich, Nicole Pamme, and Dimitri Iossifidis
What is the main focus of Week 11 in the course schedule?
Mass Spectrometry-1
What did Swedish chemist JJ Berzelius suggest about the substances in milk and eggs?
They should be called proteins
What type of bonds stabilize α-helix structure in peptide chains?
Hydrogen bonds
What is the primary stabilizing force in β-pleated sheet structure?
Hydrogen bonds
In which protein structure is the polypeptide chain reversed in direction?
Secondary structure
What type of bonds are involved in forming tertiary protein structure?
Covalent bonds
What technique is used to study different levels of protein structure by measuring the absorption of circularly polarized light?
Circular dichroism
What is the primary aim of protein purification?
To isolate a specific protein from a mixture
What is the wavelength at which proteins are detected in the Lowry method?
500-750 nm
Which protein quantification method is known for being 100 times more sensitive than the Biuret method?
BCA method
What type of electrophoresis separates proteins based on their net charge and size?
SDS-PAGE
Who pioneered protein sequencing and determined the sequence of bovine insulin?
Frederick Sanger
What gel staining method incorporates SDS to fully denature proteins and dissociate them?
SDS-PAGE
What is the purpose of dye binding methods like the Bradford protein assay?
To form a blue color complex detected at 595 nm
Which method of protein extraction is specifically mentioned as being hand-operated or motor-driven?
Blenders
What is the purpose of using detergents in protein extraction?
To break hydrophobic interactions
Which substance is recommended to protect thiol groups from oxidation during protein purification?
Mercaptoethanol
What is the purpose of using chelators in protein purification?
To stabilize proteins
What is the primary factor that influences protein precipitation using organic solvents?
Reducing water activity
Which chromatographic step involves considerations for sample volume, protein concentration, and efficiency of separation methods?
Affinity chromatography
What is the purpose of using detergents in protein extraction?
To break hydrophobic interactions
What is the function of reducing agents like DTE, DTT, and mercaptoethanol during protein purification?
To protect thiol groups from oxidation
How does ammonium sulphate salting out depend on protein properties?
It depends on protein hydrophobicity and competition for water molecules
What is the purpose of using chelators in protein purification?
To stabilize proteins and remove heavy metal ions
What is the primary function of protein separation by gel filtration chromatography?
Separating proteins based on size
How do organic solvents induce protein precipitation?
By reducing water activity and breaking protein intramolecular interactions
Study Notes
Protein Purification Techniques and Considerations
- Protein extraction methods include hand-operated or motor-driven blenders, ultrasound, vibrating bead mill, and Manton-Gaulin homogenizer
- Factors to consider in extraction medium include temperature (2-8°C) and time, with mild, moderate, and vigorous techniques available
- Extraction medium considerations include buffer salts, pH, detergents, reducing agents, chelators, proteolytic inhibitors, and bacteriostatics
- Detergents are used to break hydrophobic interactions, with concentrations below critical micelle concentration recommended
- Reducing agents like DTE, DTT, and mercaptoethanol protect thiol groups from oxidation during purification
- Chelators and metal ions may stabilize proteins but heavy metal ions can enhance oxidation; EDTA can be used to remove heavy metals
- Proteolytic inhibitors and bacteriostatics are used to prevent protein degradation and bacterial growth, respectively
- Protein precipitation methods include salting out with ammonium sulphate, organic solvents, and dialysis through semi-permeable membranes
- Ammonium sulphate salting out depends on protein hydrophobicity and competition for water molecules
- Organic solvents induce protein precipitation by reducing water activity and breaking protein intramolecular interactions
- Dialysis is used to separate proteins from small molecules and remove salts and ammonium sulphate
- Protein separation by chromatographic steps includes gel filtration, ion exchange, and affinity chromatography, with considerations for sample volume, protein concentration, and efficiency of separation methods
Test your knowledge of protein quantification, analysis, and purification techniques with this quiz. Explore methods such as UV absorption, electrophoresis, protein sequencing, and extraction techniques including protein precipitation and chromatography. Gain insights into factors to consider in extraction medium, detergents, reducing agents, and chromatographic separation methods.
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