Fibronectin (FN), a large, modular glycoprotein encoded by the?FN1?gene, is a key component of the extracellular matrix (ECM) and plasma. Synthesized primarily by fibroblasts, hepatocytes, and endothelial cells, it exists in two major forms: soluble plasma FN and insoluble cellular FN. FN mediates critical cellular processes via its integrin-binding RGD motif, including cell adhesion, migration, proliferation, and differentiation. It also participates in ECM assembly, wound healing, and angiogenesis by bridging cells to other ECM components like collagen, fibrin, and proteoglycans. Dysregulated FN expression or function is linked to pathological conditions such as fibrosis, cancer metastasis, and cardiovascular diseases, making it a vital target for biomedical research. FN binds THBS1, a secreted glycoprotein, to modulate ECM stability and regulate cellular adhesion signaling pathways.Briefly, THBS1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FN-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-THBS1 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately.Measured by its binding ability in a functional ELISA. When Recombinant FN is immobilized at 2 μg/mL(100 uLwell), the concentration of THBS1 that produces 50% optimal binding response is found to be approximately 0.054 μg/mL.