F-Box And WD Repeat Domain Containing Protein 7 (FBXW7), also known as FBW7 or CDC4, serves as a crucial component of the SCF E3 ubiquitin ligase complex. This tumor suppressor protein plays a pivotal role in maintaining cellular homeostasis by targeting several key oncoproteins for degradation. Through its ubiquitin ligase activity, FBXW7 regulates the stability of critical cell cycle regulators and signaling molecules including c-Myc, Cyclin E, Notch, and mTOR. The protein exists in three distinct isoforms that differ in their subcellular distribution and functional specialization. In human cancers, FBXW7 ranks among the most frequently mutated genes, with its inactivation observed across diverse malignancies such as colorectal carcinoma, T-cell acute lymphoblastic leukemia, and breast cancer. Besides,Ring Box Protein 1 (RBX1) has been identified as an interactor of FBXW7, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant mouse FBXW7 and recombinant human RBX1 . Briefly, biotin-linked FBXW7 were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to RBX1-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 recombinant mouse FBXW7 and recombinant human RBX1 was shown in Figure 1, the EC50 for this effect is 2.20 µg/mL.