O-Linked-N-Acetylglucosamine Transferase (OGT) is a key enzyme that catalyzes the addition of N-acetylglucosamine (O-GlcNAc) to serine or threonine residues of target proteins via O-glycosylation. This post-translational modification plays a critical role in cellular processes such as signal transduction, transcription regulation, and stress response. OGT is highly conserved across species and is essential for cell viability. Dysregulation of OGT activity has been linked to metabolic disorders, neurodegenerative diseases, and cancer. The enzyme's ability to modulate protein function dynamically, akin to phosphorylation, highlights its importance in maintaining cellular homeostasis.Besides,Ubiquitin Associated Protein 1 (UBAP1) has been identified as an interactor of OGT, thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human OGT and recombinant human UBAP1 . Briefly, biotin-linked OGT were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100μl were then transferred to UBAP1-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 human OGT and recombinant human UBAP1 was shown in Figure 1, the EC50?for this effect is 0.21 μg/mL.