This article describes the enzyme-linked immunosorbent assay (ELISA) technique for detecting protein-protein interactions. The method involves immobilizing a bait protein on a microtiter plate and subsequently adding a prey protein to form a complex, which is then quantified.
The physical interaction between two protein partners modulates downstream biological mechanisms.
To detect protein-protein interactions using the enzyme-linked immunosorbent assay, ELISA, take a multi-well immunoassay plate. Add a coating buffer containing one of the interacting proteins ― the bait protein ― and incubate. The bait proteins get adsorbed onto the well surface via non-specific hydrophobic interactions.
Discard the spent solution and wash the plate with a buffer to remove unbound proteins. Add a blocking solution and incubate, allowing the solution's blocking agent molecules to block non-specific binding sites on the well's surface.
Add the interacting protein partner ― the prey protein ― tagged with histidine residues for easy quantification. Incubate, allowing the prey protein to specifically bind to its interactive partner ― the bait. Add a solution of anti-histidine antibodies conjugated with horseradish peroxidase enzymes that bind to the prey, forming a bait-prey-antibody complex.
Pipette a solution containing the enzyme's substrate and hydrogen peroxide and incubate.
The horseradish peroxidase enzyme uses hydrogen peroxide for the catalytic oxidation of its substrate ― forming a blue-colored product. Add an acidic solution that denatures the enzyme to stop the reaction, forming a yellow reaction product.
Using a microplate reader, measure the product's color intensity, which is proportional to the amount of the bound prey protein and indicates successful protein-protein interaction.
To prepare for ELISA, dispense 100 microliters of protein solution into each well of a Polysorp microtiter plate. Close the plates with the lid, and store at 4 degrees Celsius overnight to facilitate the immobilization of protein onto microtiter plate wells. Discard the solution from the microtiter plate by tilting upside down over the sink. Then, wash the wells three times with 300 microliters of PBS per well.
Block the coated wells for 1 hour at room temperature with PBS, containing 2.5 weight volume percent BSA. After removing the blocking solution, wash the wells three times with 300 microliters of PBST per well. Now, add 100 microliters of 50 nanomolar recombinant His-tagged PH to each sample. In the control wells, add only 100 microliters of PBS-BSA. Incubate the plate for 1 hour at room temperature.
Following incubation, discard the protein solution and remove the unbound proteins by washing the wells three times with 300 microliters of PBST per well. Then, add 100 microliters of PBS-BSA, containing horseradish peroxidase-conjugated anti-His polyclonal antibodies.
After incubating for 1 hour at room temperature, wash the wells three times with 300 microliters of PBST per well. Detect the antigen-antibody complexes by adding 100 microliters of ELISA detection reagent to each well. Then, add 50 microliters of 1 molar sulfuric acid per well to stop the reaction. Measure the optical density of the wells at 450 nanometers, using a microplate reader.
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