ELISA-Based Method to Analyze Interactions between Bait and Prey Proteins
Trascrizione
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.
