Mothers Against Decapentaplegic Homolog 2 (Smad2) is a pivotal intracellular mediator of the TGF-β signaling pathway that regulates diverse cellular processes including proliferation, differentiation, and apoptosis. As an R-Smad protein, Smad2 transduces signals from activated TGF-β receptors to the nucleus, where it modulates gene expression in complex with Smad4. Smad2 is essential for embryonic development and tissue homeostasis, with its dysfunction linked to cancer and fibrotic diseases. Following TGF-β stimulation, Smad2 binds to and is phosphorylated by the activated Transforming Growth Factor Beta Receptor I (TGFbR1), initiating downstream signaling cascades.To detect the activity of recombinant Smad2, a functional ELISA assay was performed to evaluate the interaction between recombinant human Smad2 and recombinant human TGFbR1.Briefly, biotin-linked Smad2 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to TGFbR1-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST 3 times and incubation with Streptavidin-HRP for 30min, then wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50µl stop solution to the wells and read at 450nm immediately. The binding activity of Smad2 and TGFbR1 was shown in Figure 1, the EC50 for this effect is 0.77µg/mL.