Lecture 10: Protein Characterization

Question 1

Studying Biomolecular Interactions

Answer 1

• Protein-protein interactions: Co-immonuprecipitation, cross-linking reagents, FRET
• Protein-molecule interactions: transport assays, enzymatic reactions
• Enzymes and fluorescent proteins can be used to generate a visual response

Question 2

Pull-Down Assays

Answer 2

• Pull down X with and see if Y comes along
• Use antibody specific to X and Y

Question 3

Capture complexes with cross-linking

Answer 3

• Chemical cross-linking can form covalent bonds between molecules
• Cross-linking studies can be used to reveal inter and intra molecular organization of amino acids
• Formaldehyde is example of non-specific chemical cross-linker

Question 4

Chemical cross-linkers

Answer 4

• Disulfide bond formation is an example of cross-linking that can occur between cysteines in the presence of oxygen

Question 5

Assessing Interactions

Answer 5

• Purification tags can assist with separation of complexed from other non-specific binding proteins
• Chemical crosslinking is useful for stabilizing structures for x-ray crystallography

Question 6

BioID

Answer 6

• BioID is a proximity-dependent biotinylation assay that can be used to identify protein-protein interactions in cells
• BirA is a biotin protein ligase that is fused to a protein of interest and will biotinylate any proximity proteins
• Biotinylated proteins can be purified using streptavidin beads and identified using WB or mass spec