Lecture 10 Protein Characterization Flashcards
What factors should be considered when designing an experiment to study proteins? (5)
Temperature, pH, detergents, cofactors, substrates.
What are the two main environments for protein experiments?
In vitro (test tube) and in vivo (live cells).
What is the purpose of control experiments in protein studies?
To ensure accurate interpretation of data and results.
Name two methods for studying protein-protein interactions.
Co-immunoprecipitation and pull-down assays.
What is FRET used for in protein studies?
It is used to study protein-protein interactions via energy transfer.
What is the challenge with non-covalent protein interactions?
They may be short-lived.
What reporter proteins are commonly used to visualize protein interactions?
Luciferase and β-galactosidase.
How does a pull-down assay work?
It uses a special molecule to pull down a protein and detect if another protein binds to it.
What is used to detect bound proteins in a pull-down assay?
Western blot.
What is cross-linking used for in protein studies?
To form covalent bonds between molecules and stabilize complexes.
Give an example of a chemical cross-linker.
Formaldehyde
What functional groups can be targeted in cross-linking reactions? (4)
Amines, carboxyls, carbonyls, sulfhydryls.
What is BioID used for and how does it work?
To identify protein-protein interactions by biotinylating nearby proteins.
How close must proteins be for BioID to biotinylate them?
Within ~10 nm
What techniques can you use to detect protein interactions if you only have an antibody for Protein A? (4) BCFP
BioID, chemical cross-linking, fluorescence microscopy, pull-down assay.
