Split Luciferase Complementation Assay to Identify Specific Protein-Protein Interactions
Transcrição
Protein-protein interactions are crucial for vital cellular functions.
To study the interactions between transmembrane proteins — membrane proteins that span the lipid bilayer of the cell membrane, begin with the wells of a multi-well plate containing an adherent mammalian cell culture.
Next, add a solution containing a suitable transfection agent and a pair of expression plasmids.
One of the expression plasmids encodes a transmembrane protein of interest fused to the small non-functional fragment of the luciferase enzyme; the other plasmid encodes a second transmembrane protein of interest fused to the large non-functional fragment of the luciferase enzyme.
Incubate the plate. The transfection agent facilitates the cellular uptake of the expression plasmids.
Successfully transfected cells express the transmembrane proteins. The interaction between the expressed transmembrane proteins brings the small and large fragments facing the cytoplasm into close proximity, causing them to bind and form the active luciferase enzyme.
Replace the media in the wells with serum-free media to minimize background luminescence. Add furimazine — a cell-permeable luciferase substrate — solution. Furimazine enters the cells, following which luciferase oxidizes the furimazine, generating high-intensity luminescence.
Using a microplate reader, measure the luminescence, which is suggestive of the strong interaction between the transmembrane proteins.
