A High-Throughput Solid-Phase Microplate Protein-Binding Assay to Investigate Interactions between Myofilament Proteins
Figure 1
Solid-phase protein-binding assay method. (a) Binary binding assay to quantify the interaction between an immobilized protein and an interacting soluble protein. (1) The immobilized protein of interest is maximally coated into the wells of a 96 well assay plate. (2) Unbound protein is removed by washing, and remaining surfaces blocked with a nonspecific protein. (3) The plate is washed, and binding is evaluated by incubation with serial dilutions of a soluble interacting protein. (4) Following another wash to remove unbound interacting protein, bound protein is detected by ELISA with a primary antibody against the interacting protein, a HRP-conjugated second antibody and substrate development to quantify bound protein by absorbance. (b) Multilayer binding assay to quantify the interaction between an immobilized protein and two related interacting soluble proteins. The multilayer protein binding is conducted similar to the binary binding assay in (a) with the exception that, following binding of the first soluble protein and washes, the wells are incubated with another protein that binds the first protein bound to the immobilized protein followed by ELISA quantification. (c) Competitive binding assay to quantify the interaction between an immobilized protein and an interacting soluble binding protein in the presence of varied amounts of a second binding protein. Protein coating and blocking are conducted as in (a) and (b) with the exception that during the soluble protein binding step, addition of the first interacting protein of interest is conducted at a constant concentration in the presence of serial concentrations of a competitive binding protein. Resultant binding of the constant concentration of the first binding protein of interest to the immobilized protein is then quantified by ELISA with a specific antibody. ELISA quantification of the degree of competition will be detected by decreases in the final reading of absorbance.
