Human Integrin Beta 3 (ITGb3), a critical transmembrane glycoprotein subunit encoded by the?ITGB3?gene, is abundantly expressed in human platelets, endothelial cells, osteoclasts and tumor cells. As a core subunit of integrin heterodimers, human ITGb3 non-covalently couples with specific alpha subunits to mediate cell adhesion, migration, proliferation and survival by binding extracellular matrix (ECM) proteins such as fibrinogen and vitronectin. Its activation is stringently modulated by intracellular signaling pathways, and the dysregulation of human ITGb3 is closely associated with multiple pathological conditions including thrombotic disorders, cancer metastasis and osteoporosis. In human platelets, the ITGb3-containing αIIbβ3 heterodimer serves as a central receptor for fibrinogen cross-linking, which is indispensable for platelet aggregation and blood clot formation; in tumor biology, it promotes angiogenesis and tumor cell invasion through interactions with the tumor microenvironment. Human ITGb3 physically binds to Rat ITGa5 to form the α5β3 heterodimer, which enhances cell adhesion to fibronectin in the ECM.Briefly, ITGa5 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ITGb3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ITGa5 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 ITGb3 is lmmobilized at 2 μg/mL(100 uLwell), the concentration of ITGa5 that produces 50% optimal bindingresponse is found to be approximately 4.89 μg/mL.