Enzyme Assays

Pulldown Assays

• Requires antibodies specific to proteins of interest
• Protein specific antibodies are bound to beads
• cells are broken open and incubated with beads
• beads are washed to remove non-interacting proteins

• After isolating your antibody specific protein, run a western blot using antibodies specific for potential interaction proteins.
• You can also run mass spectrometery to identify unknown proteins

Cross-linking

• Protein complex can be analyzed via cross-linking
• shows inter- and intra- molecular organization based on location
• Cross-linking is the formation of covalent bonds between molecules
• Primary amines
• Carboxyles
• Carbonyls
• Sulfhydryls
• Formaldyhyde is often used as a non-specific chemical cross-linker
• Cross-linkers can also have affinity tags such as biotin (streptavidin affinity chromatography)
• Spacers can be incorperated into cross-linkers to provide flexibility in range of interaction
• Purification and uses:
• Purification tags
• SDS-PAGE
• Western blots
• Mass spectrometery
• X-ray crystallography

Two-Hybrid System

• Two potential interaction proteins can be made fusion proteins with bait and prey subunits
• When these proteins come together a reporter is activated allowing detection of interaction
• The reporter can even bind DNA and allow for transcription of a gene upon activation

BioID

• Allows for protein interactions to be observed through a biotinylation assay
• BirA is fused onto a protein of interest and will biotinylate any interacting proteins (within 10 nm)
• The results can then be monitored via western blot or mass spec

Fluorescence Based Assays

• Fluorescence/Förster Resonance Energy Transfer
• A donor fluorophore emits light that excites a close acceptor fluorophore

• Bioluminescence Resonance Energy Transfer
• A enzyme reaction results in the donor producing light

• Can be used to analyze FRET and BRET assays
• Can also be used to co-localize proteins that have been fluorescently tagged
• Can show cellular localizatin, but is not a way of identifying interactions