Sex Determining Region Y Box Protein 9 (SOX9) is a transcription factor belonging to the SOX (SRY-related HMG-box) family, which plays crucial roles in embryonic development, cell differentiation, and tissue homeostasis. It is essential for chondrogenesis, male sex determination, and the maintenance of various progenitor cell populations. SOX9 regulates the expression of target genes by binding to specific DNA sequences, influencing pathways such as Wnt/β-catenin and TGF-β.Besides,Fibroblast Growth Factor 9 (FGF9) has been identified as an interactor of SOX9, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse SOX9 and recombinant human FGF9. Briefly, SOX9 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FGF9-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-SOX9 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. The binding activity of recombinant mouse SOX9 and recombinant human FGF9 was shown in Figure 1, the EC50?for this effect is 0.08μg/mL.