Glycosyltransferase Activity, BioAssay(TM) Kit

Supplier:

United States Biological

Cat no: G8183-01

Glycosyltransferase Activity, BioAssay(TM) Kit

Test Principle:\nThe 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.\n\nKit Components:\n1. *Coupling Phosphatase 1 (100ng/ml) NS0-expressed recombinant human ENTPD3/CD39L3 in 20mM Tris, 120mM NaCl, 4mM CaCl2, 20% glycerol, pH 7.5, 1x200ml \n2. Phosphatase Buffer 1 (10X) 250mM Tris, 100mM CaCl2, pH 7.5, 1x1.5ml \n3. *UDP (5mM) in 10mM sodium borate, pH 9.0, 1x100ml \n4. MnCl2 (100mM), 2x1.5ml \n5. Phosphate Standard (1mM KH2PO4), 1x 200ml \n6. Malachite Green Reagent A in 3M sulfuric acid, 3x3ml\n7. Malachite Green Reagent B (malachite green oxalate and polyvinyl alcohol), 3x3ml\n\nThe protocol presented is for a 96-well format. Materials provided are sufficient for two 96-well microplates or equivalent. \n\nStorage and Stability:\n*Store Coupling Phosphatase 1 and UDP at at -20 degrees 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.

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SPECIFICATIONS

Catalog Number

G8183-01

Size

1Kit

References

1. Wu, Z.L. et al. (2011) Glycobiology [epub ahead of print].

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SUPPLIER INFO

United States Biological

Address:

4 Technology Way, Salem, MA 01970, US

Phone:

+1 800 520 3011

Website:

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