Beta-1,4-galactosyltransferase 1 (B4GALT1) is a key glycosyltransferase. It mainly mediates the addition of galactose to glycoproteins and glycolipids in the Golgi apparatus, playing essential roles in cell adhesion, signaling, and normal physiological functions.The galactosyltransferase B4GALT1 can transfer the galactosyl group from UDP-galactose to N-acetyl-α-D-glucosamine. The resulting UDP can generate free phosphate under the action of a coupled phosphatase. Dilute UDP-galactose, coupling phosphatase 1, and GlcNAc in assay buffer, then mix equal volumes to prepare the reaction mixture. Dilute rhB4GalT1 in assay buffer. Prepare a 100?μM phosphate standard stock and perform six half serial dilutions for a 0.078–5.0?nmol/well curve. Add 50?μL standards and blank to the plate. Add 20?μL enzyme or buffer control, then start the reaction with 30?μL reaction mixture. Incubate 15?min at room temperature. Add malachite green reagents A and B with water sequentially, mix, incubate 20?min, and read absorbance at 620?nm.The specific activity of recombinant human B4GALT1 is > 27000 pmol/min/μg.Specific Activity (pmol/min/μg) = [Phosphate released (nmol) × 1000 (pmol/nmol)] / [Incubation time (min) × Amount of enzyme (μg)]<br/>?*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.