Carbohydrate Sulfotransferase 5 (CHST5) is a key Golgi-resident sulfotransferase enzyme. It specifically catalyzes the transfer of sulfate groups from 3′-phosphoadenosine-5′-phosphosulfate to carbohydrate moieties on glycoproteins and glycolipids, predominantly acting on N-acetylglucosamine residues within mucin-type O-glycans. Expressed heavily in intestinal epithelial tissues, it shapes the sulfated glycan landscape of the intestinal mucosal barrier. Deficient CHST5 activity disrupts mucosal glycosylation, weakens gut protective functions, and correlates with inflammatory bowel disorders, altered microbiota adhesion, and impaired epithelial defense. Its sulfation modification modulates glycoprotein stability, ligand recognition, and cell-surface signaling to sustain intestinal homeostasis. CHST5 mediates sulfation modification of MUC2 glycans to stabilize secreted intestinal mucus layers. Briefly, MUC2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μL were then transferred to CHST5-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-MUC2 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 human CHST5 is immobilized at 2 μg/mL(100 μL/well), the concentration of MUC2 that produces 50% optimal binding response is found to be approximately 1.918 μg/mL.