Lecture 23 Flashcards
What is SDS-PAGE and how is it performed?
Analytical method for the separation of charged molecules in mixtures by their molecular masses in an electric field
Denatured proteins are loaded onto a gel of polyacrylamide, which is placed in an electrophoresis buffer with suitable electrolytes; thereafter, a voltage is applied, which causes a migration of negatively charged molecules through the gel in the direction of the anode (+ pole)
What is 2D-PAGE and how is it performed?
IEF (isoelectric focusing) tube gel is denatured and laid on top of an SDS-PAGE and electrophoresed again
A mixture of proteins is separated by charge in one dimension and by mass in a second dimension, producing a series of spots on a gel
What is mass spec and how is it performed?
Analytical technique that ionizes chemical species and sorts the ions based on their mass-to-charge ratio; measures the masses within a sample
Solid, liquid, or gas sample is ionized, which may cause some of the samples molecules to break into charged fragments; these ions are then separated according to their mass-to-charge ratio, by accelerating them and subjecting them to an electric or magnetic field; ions of the same mass-to-charge ratio will undergo the same amount of deflection
What is the Yeast 2 hybrid technique?
Molecular technique used to discover protein-protein interactions and protein-DNA interactions by testing for physical interactions (binding) between 2 proteins or a single protein and a DNA molecule
What is a Western Blot and how is it performed?
- Extract proteins
- Electrophorese
- Transfer to membrane
- Detect with protein specific antibodies
What are protein microarrays and how are they performed?
Can be used to study protein-protein interactions of thousands of proteins at once; primarily used for diagnostics, protein expression profiling, protein functional analysis, antibody characterization, and treatment development
You have one control sample and one experimental sample; you incubate samples with array membrane, then incubate with biotin-labeled anti-cytokine antibodies, then incubate with streptavidin-HRP, followed by chemiluminescence detection; differential signals correspond to differences between the two samples in cytokine activities
What is affinity capture and how is it performed?
Method of separating biochemical mixtures based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand
Proteins specifically bind to the column, the unbound proteins are washed away, and then the elution of the specifically bound proteins
Define the interactome/reactome
Interactome: whole set of molecular interactions in a particular cell; refers to physical interactions among molecules, but can also describe sets of indirect interactions among genes
Reactome: Overview of pathways involved in protein metabolism; metabolism of proteins covers the full life cycle of a protein from its synthesis to its post-translational modification and degradation
What is the importance of post-translational modifications?
Refers to the covalent and generally enzymatic modification of proteins following protein biosynthesis; this increases protein diversity
What are prions? What are tau-plaques?
Prions: infectious agents composed entirely of a protein material that can fold in multiple, structurally abstract ways, at least one of which is transmissible to other prion proteins, leading to disease in a manner that is epidemiologically comparable to the spread of viral infection (ex: Creutzfeldt-Jakob disease)
Tau-Plaques: Can lead to alzheimer’s disease
What are the basic tools used to determine the significance of mutations (AA changes)?
SIFT: predicts whether an AA substitution affects protein function
PolyPhen-2: predicts possible impact of an AA substitution on the structure and function of a human protein using straightforward physical and comparative considerations
What are the examples of protein prediction softwares?
Phyre: used for protein structural prediction
Uniprot: database of protein functions
Describe globular, fibrous, and membranous proteins
Globular: almost all are soluble and many are enzymes
Fibrous: often structural, such as collagen
Membranous: often serve as receptors or provide channels for polar or charged molecules to pass through the cell membrane
What is diagnostics?
Involves detection of biomarkers: antigens and antibodies in blood samples; monitoring of disease states and response therapy; monitoring of environment and food
What is protein expression profiling?
Identifies variation in quantities of proteins in extracts from tissues or cells
What is protein functional analysis?
Identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, enzymatic substrates, and receptor ligands
What is antibody characterization?
Examines cross-reactivity, antibody specificity, and antibody epitope mapping
What is treatment development?
Involves the design of antigen-specific therapies for autoimmune disease, cancer, and allergies; identification of small molecule targets that could potentially be used as new drugs
