Recombinant Mouse alpha3GalT/GGTA1 Protein, CF Summary
Trp80-Val394, with C-terminal 6-His tag
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CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35.|
|Shipping||The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 50 mM MES, 20 mM MnCl2, pH 7.0
- Recombinant Mouse alpha 3GalT/GGTA1 (rmGGTA1) (Catalog # 7887-GT)
- UDP-Galactose (Sigma, Catalog # U4500), 10 mM stock in deionized water
- alpha -Lactose (Sigma, Catalog # L2643), 0.3 M stock in deionized water
- Glycosyltransferase Activity Kit (Catalog # EA001)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Universal Sulfotransferase Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.
- Prepare standard curve by performing six one‑half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare reaction mixture containing 0.8 mM UDP‑Galactose, 8 µg/mL Coupling phosphatase 1, and 40 mM alpha -Lactose in Assay Buffer.
- Dilute rmGGTA1 to 1.2 µg/mL in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
- Load 25 µL of the 1.2 µg/mL rmGGTA1 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 µL of deionized water to all wells.
- Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Phosphate released* (nmol) x (1000 pmol/nmol)|
|Incubation time (min) x amount of enzyme (µg)|
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.Per Reaction:
- rmGGTA1: 0.03 µg
- Coupling phosphatase 1: 0.2 µg
- UDP-Galactose: 0.4 mM
- alpha -Lactose: 20 mM
GGTA1, also designated as alpha 3GalT, is an alpha -1,3 galactosyltransferase that catalyzes the transfer of galactose from UDP-alpha -D-galactose to various glycoconjugates to form non‑reducing terminal alpha -1,3-linked galactosyl moieties (1). This enzyme is expressed in many mammalian species but is absent from humans, apes, and Old World monkeys due to the mutational inactivation of the gene (2). Therefore, humans do not have galactosyl alpha -1,3-galactosyl structures on glycoconjugates and produce large amounts of antibody to this alpha -Gal epitope. The production of this endogenous antibody results from exposure to intestinal bacteria and other antigens (3). GGTA1 is of medical interest because the immune responses may be enhanced when selected targets are decorated with galactosyl alpha -1,3-galactosyl structures (4). Additionally, the presence of anti-alpha -Gal antibodies is a barrier to xenotransplantation of organs, therefore, genetic manipulation of the GGTA1 gene and the development of inhibitors of the enzyme are of interest as complementary approaches to reduce rejection of xenotransplanted organs. Structurally and mechanistically GGTA1 is a model for several homologous glycosyltransferases that differ in donor and acceptor substrate specificity, including the histo blood group A and B glycosyltransferases, Forssman glycolipid synthase, and isogloboside 3 synthase (5). The activity of the recombinant GGTA1 is measured using a phosphatase-coupled glycosyltransferase assay (6).
- Larsen RD, et al.(1989) Proc. Natl. Acad. Sci. U. S. A. 86:8227.
- Joziasse, D.H. et al. (1992) J. Biol. Chem. 267:5534.
- Wigglesworth KM, et al. (2011) J. Immunol. 186:4422.
- Dequchi, T. et al. (2010) Cancer Res. 70:5259.
- Breton, C. et al. (2005) Glycobiology 16:29R.
- Wu, Z.L. et al. (2011) Glycobiology. 21:727.
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