Test Principle:The Glycosyltransferase Activity Kit provides a simple, non-radioactive high-throughput compatible format for assaying the enzyme activity of all glycosyltransferases that use diphosphonucleotide sugars as donor substrates. This kit takes advantage of nucleotidase ENTPD3/CD39L3 as a coupling phosphatase to remove inorganic phosphate quantitatively from the leaving nucleotide diphosphate, such as UDP or GDP, of glycosyltransferase reactions (Figure 1). The released inorganic phosphate is then detected by Malachite Green phosphate detecting reagents. The amount of inorganic phosphate released by the coupling phosphatase is equal to the nucleotide sugar consumed or glycoconjugate product generated; therefore, the rate of inorganic phosphate production reflects the kinetics of a glycosyltransferase reaction.
Kit Components:1. *Coupling Phosphatase 1 (100ng/ml) NS0-expressed recombinant human ENTPD3/CD39L3 in 20mM Tris, 120mM NaCl, 4mM CaCl2, 20% glycerol, pH 7.5, 1x200ml 2. Phosphatase Buffer 1 (10X) 250mM Tris, 100mM CaCl2, pH 7.5, 1x1.5ml 3. *UDP (5mM) in 10mM sodium borate, pH 9.0, 1x100ml 4. MnCl2 (100mM), 2x1.5ml 5. Phosphate Standard (1mM KH2PO4), 1x 200ml 6. Malachite Green Reagent A in 3M sulfuric acid, 3x3ml7. Malachite Green Reagent B (malachite green oxalate and polyvinyl alcohol), 3x3ml
The protocol presented is for a 96-well format. Materials provided are sufficient for two 96-well microplates or equivalent.
Storage and Stability:*Store Coupling Phosphatase 1 and UDP at at -20°C. Store other components at RT. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
