This article details a method for studying protein-protein interactions between transmembrane proteins using luminescence. The approach involves transfecting mammalian cells with expression plasmids and measuring luminescence as an indicator of protein interactions.
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.
Harvest the adherent HEK293T cell culture by trypsinization, and re-suspend the cells in a dedicated complete growth medium. Plate the cells onto a clear bottom, white side, 96-well plate. Adjust the total number of wells to accommodate all tested combinations and controls, including replicates. Then, culture the cells overnight in standard conditions.
On the next day, transfect the cells with the desired combinations of expression plasmids. Dilute the plasmids in a serum-free medium to 6.25 nanograms per microliter for each construct, and add the lipid-based transfection reagent at an appropriate lipid-to-DNA ratio.
Incubate the plasmids according to manufacturer's instruction. Then, add 8 microliters of the lipid-DNA mixture to the designated wells. Mix the content of the plate with gentle rotation, and culture the cells for 20 to 24 hours in standard conditions.
On the next day, replace the conditioned medium in each well with 100 microliters of a serum-free medium, making sure that the cells do not detach upon medium exchange.
Just before measuring luminescence, mix one volume of furimazine with 19 volumes of a dilution buffer. Add 25 microliters of the furimazine working solution into each well, and gently mix the plate by hand, or with an orbital shaker.
Insert the plate into a luminescence microplate reader, and equilibrate the plate at 37 degrees Celsius for 10 to 15 minutes. Select the wells to be analyzed, and read luminescence with an integration time of 0.3 seconds. Continue to monitor luminescence for up to two hours when required.
繁體中文
