30 Questions
What is the main principle behind electrophoresis?
Separation of macromolecules from complex mixtures by application of an electric field
What is the purpose of assays to determine protein concentration?
To normalize several biological parameters per unit of proteins
Which method relies on changes in the color of a compound upon binding to proteins to determine protein concentration?
Lowry assay
What is the significance of a darker purple color in the BCA assay reaction?
Indicates a higher protein concentration
How are macromolecules in a mixture separated during electrophoresis?
They migrate at different speeds based on the nature of the gel and their characteristics
What is the purpose of comparing color changes in a spectrophotometric plate reader during a total protein assay?
To deduce the protein concentration of the sample
What is the main purpose of using Fluorescence Resonance Energy Transfer (FRET) in protein studies?
To detect interactions and measure distance between two proteins
What happens if two fluorophores are distanced efficiently in FRET?
No fluorescence is emitted
Why is labeling two proteins that are already expected to interact not a good tool to discover new interactions using FRET?
It does not allow for the discovery of new interactions
In the context of FRET, what does the emission of fluorescent light by one fluorophore do to the other fluorophore?
It excites the other fluorophore, causing it to emit fluorescent light too
What is the role of Fluorescence Resonance Energy Transfer (FRET) in detecting change in protein conformation?
It can measure distance between two interacting proteins based on fluorescence intensity
What can FRET be used to detect in live cells over time?
Time and space resolved protein-protein interaction between specific proteins
What is the main purpose of two-dimensional gel electrophoresis (2D-PAGE)?
To separate proteins based on their isoelectric point and molecular weight
What can cause molecular supercomplexes to dissociate, leading to less effective respiratory chain and ATP depletion syndromes?
Disease
Which technique is used to analyze protein modifications that cannot be detected by SDS-PAGE?
Isoelectric focusing (IEF)-PAGE
What is the main limitation of the Sanger method for protein sequencing?
It required large amounts of purified protein for sequencing
How are proteins identified in cellular homogenate mixtures before extraction and digestion with trypsin?
By first separating them using 1D or 2D PAGE
Which technique involves cleaving proteins between Lysine and Arginine residues to generate peptides for mass spectrometry analysis?
Trypsin digestion
Which method can be used to study protein-protein interactions by measuring the distance-dependent transfer of energy from a donor molecule to an acceptor molecule?
Fluorescence Resonance Energy Transfer (FRET)
What is the traditional approach for solving the tertiary structure of proteins?
X-Ray crystallography
Which method involves biotinylating proteins that come close to the protein of interest to track interaction partners of a protein?
BioID method
Which method is used to sequence peptide fragments and compare peptide masses with a protein database for protein identification?
Tandem MS/MS
What is the artifact that can occur in Co-immunoprecipitation involving non-specific binding and unspecific proteins stuck to beads, requiring controls to distinguish genuine interactions?
Non-specific binding
Which method examines the relative abundance of peptide masses to identify proteins, requiring nano-pico mole amounts of the protein?
Peptide mass fingerprinting
What initiates the chemical crosslinking of Acrylamide and Bisacrylamide in PAGE gels?
Ammonium persulphate and TEMED
Which variant of PAGE linearizes proteins and destroys all chemical/covalent links, allowing proteins to become negatively charged and migrate based on size?
SDS-PAGE
Which type of PAGE preserves protein-protein interactions that exist in vivo and allows recovery of proteins in their functional form, with retained enzyme activity?
Native PAGE
What does 2-Dimensional PAGE involve?
Two successive rounds of electrophoresis
What can be added to the gel in PAGE to detect the presence of a specific protein in a sample?
Specific antibodies
What is used in PAGE to compare migration distances of proteins of interest to molecular weight ladders and estimate their apparent molecular weight based on the migration distance?
Molecular weight ladders
Study Notes
Polyacrylamide Gel Electrophoresis (PAGE) in Biomedicine
- Polyacrylamide Gel Electrophoresis (PAGE) is a significant advancement in protein resolution and characterization.
- PAGE can be performed using various matrices such as paper and different gels for protein separation.
- PAGE gels are formed using chemical crosslinking of Acrylamide and Bisacrylamide, initiated by ammonium persulphate and the base TEMED, solidifying in 10-15 minutes.
- The percentage of acrylamide in the gels varies based on the nature and size of the macromolecule being separated, with lower percentages used for nucleic acids and higher percentages for proteins.
- SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is the most common variant, which linearizes proteins and destroys all chemical/covalent links, allowing proteins to become negatively charged and migrate based on size.
- SDS-PAGE can be performed in reducing or non-reducing conditions to examine associations between proteins, with reducing conditions using agents that reduce disulfide bonds.
- Native PAGE preserves protein-protein interactions that exist in vivo and allows recovery of proteins in their functional form, with retained enzyme activity.
- 2-Dimensional PAGE involves two successive rounds of electrophoresis and dramatically improves separations and resolution of complex mixtures of proteins.
- Gradients can be used in PAGE to improve separation, with injection wells and changing pore sizes in the gel.
- PAGE allows the resolution of proteins in complex mixtures, estimation of their size, and determination of some of their properties.
- Specific antibodies can be added to the gel to detect the presence of a specific protein in a sample, allowing for precise measurements.
- PAGE gels can be used to compare migration distances of proteins of interest to molecular weight ladders and estimate their apparent molecular weight based on the migration distance.
Test your knowledge of Polyacrylamide Gel Electrophoresis (PAGE) in biomedicine with this quiz. Explore the different types of PAGE, its applications, and the techniques used for protein separation and characterization.
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