Product Category
Glycosyltransferase Activity Kits
A Non-radioactive Assay for Analyzing Glycosyltransferase Activity
R&D Systems offers two kits for analyzing the activity of glycosyltransferases that use activated sugar nucleotides as donor substrates. The Glycosyltransferase Activity Kit (Catalog # EA001) and the Sialyltransferase Activity Kit (Catalog # EA002) include specific phosphatases that release inorganic phosphate from nucleotides produced during glycosyltransferase reactions. The Glycosyltransferase Activity Kit includes the enzyme CD39L3 to release beta-phosphate from diphosphonucleotides, such as UDP, while the Sialyltransferase Activity Kit includes CD73, a 5'-nucleotidase that removes the phosphate from 5'-monophosphates, such as CMP. The released phosphate is then detected using colorimetric reagents. Because the concentration of released phosphate is directly proportional to the number of sugar molecules transferred during the reaction, these kits provide researchers a simple, non-radioactive method for measuring kinetic parameters of
glycosyltransferases. Furthermore, the assay is performed in multi-well plates and quantified by a plate reader, making it amenable to high-throughput screening.
Features of the Glycosyltransferase & Sialyltransferase Activity Kits
Versatile
- Applicable for all glycosyltransferases that generate leaving groups with a removable phosphate
Convenient
- Non-radioactive
- No time-consuming separation steps (e.g. column chromatography)
Quantitative
- Nucleotidases provided remove phosphate in a quantitative manner allowing for accurate kinetic analysis
Amenable to High-throughput Analysis
- Assay can be performed in standard multi-well microplates
- Utilizes a standard colorimetric plate reader for analysis
Available Kits
Glycosyltransferase Activity Kit (Catalog # EA001)
Sialyltransferase Activity Kit (Catalog # EA002)
Kit Contents
Please refer to product-specific datasheets for a complete list of contents for each individual assay.
- Nucleosidase
- Nucleoside Phosphate
- Phosphatase Buffer
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- 100 mM MnCl2
- Phosphate Standard
- Malachite Green Reagents A & B
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Assay Principle
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| The assay procedure for these kits involves preparing a reaction mix containing a donor molecule, acceptor substrate, and coupling phosphatase. The reaction is then initiated by adding the glycosyltransferase or sialyltransferase. Malachite Green Phosphate detection reagents are added and absorbance is read at 620 nm. |
Data Examples
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| Activity of B4GalT1. The ability of Recombinant Human beta-1,4-Galactosyltransferase 1/B4GalT1 (Catalog # 3609-GT) to transfer galactose from UDP-galactose to N-acetyl-alpha-D-glucosamine was measured using the Glycosyltransferase Activity Kit (Catalog # EA001). The activity was plotted against enzyme input, and the specific activity of B4GalT1 was determined to be 1806 pmol/min/µg. |
Activity of ST6GALNAC2. The ability of Recombinant Mouse ST6 Sialyltransferase 2/ST6GALNAC2 (Catalog # 6468-GT) to transfer Neu5Ac (sialic acid) from CMP-Neu5Ac to bovine asialofetuin was measured using the Sialyltransferase Activity Kit (Catalog # EA002). The activity was plotted against enzyme input, and the specific activity of ST6GALNAC2 was determined to be 774 pmol/min/µg. |
For more data examples generated using this novel assay method, please refer to Wu, Z.L. et al. (2010) Glycobiology 21.