Recombinant Human GAL3ST2 Protein, CF Summary
His32-Ala398 with an N-terminal 6-His tag
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 and NaCl.|
|Shipping||The product is shipped with dry ice or equivalent. 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: 25 mM MES, 15 mM MgCl2, pH 5.5
- Coupling Phosphatase Buffer: 100 mM Tris, 15 mM MgCl2, pH 8.0
- Recombinant Human Galactose‑3‑O‑sulfotransferase 2/GAL3ST2 (rhGAL3ST2) (Catalog # 7719-ST)
- Donor Substrate: 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
- Acceptor Substrate: alpha -Lactose (Sigma, Catalog # L2643), 0.3 M stock in deionized water
- Universal Sulfotransferase Activity Kit (Catalog # EA003)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard 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 a Substrate mixture of 0.8 mM PAPS and 10 mM alpha -Lactose in Assay Buffer.
- Dilute rhGAL3ST2 to 4 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 4 µg/mL rhGAL3ST2 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of Substrate mixture to the wells, excluding the standard curve.
- Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
- Dilute Coupling Phosphatase 3 (supplied in kit) to 10 µg/mL in Coupling Phosphatase Buffer.
- Add 50 µL of 10 µg/mL Coupling Phosphatase 3 to each well, excluding the standard curve. Add Coupling Phosphatase Buffer to the standard curve.
- Cover the plate with a plate sealer and incubate at 37 °C for 10 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 50 µL of deionized water to all wells. Mix briefly.
- 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.
**Use the incubation time of the reaction between the Enzyme and Substrate mixture only, exclude additional incubation times.
- rhGAL3ST2: 0.1 µg
- Coupling Phosphatase 3: 0.5 µg
- PAPS: 20 nmol
- alpha -Lactose: 250 nmol
Background: Galactose-3-O-sulfotransferase 2/GAL3ST2
Galactose-3-O-sulfotransferase 2 (GAL3ST2) is a type II Golgi resident transmembrane protein. It transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the C-3 hydroxyl group of nonreducing beta -galactosyl residues present on various glycans, such as N-acetyllactosamine, lactose, lacto-N-tetraose (Lc4), lacto-N-neotetraose (nLc4), type 1 oligosaccharides (Gal beta 1-3GlcNAc-R), type 2 oligosaccharides (Gal beta 1-4GlcNAc-R), and core 1 O-glycans (Gal beta 1-3GalNAc alpha 1-Ser/Thr) (1). Given its broad substrate specificity, GAL3ST2 is capable of radioisotope labeling of non-reducing terminal galactose on glycoproteins or glycolipids with 35S. In addition, it can be used to generate galactin-4 and galactin-8 specific ligands (2, 3). The protein is widely expressed in various tissues (1). The enzymatic activity of the recombinant human GAL3ST2 is measured using a phosphatase-coupled assay (4).
- Honke, K. et al. (2001) J. Biol. Chem. 276: 267.
- Ideo, H. et al. (2002) Glycobiology 12:199.
- Ideo, H. et al. (2011) J. Biol. Chem. 286:11346.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
Citation for Recombinant Human GAL3ST2 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
Influence of sulfonated and diet-derived human milk oligosaccharides on the infant microbiome and immune markers
Authors: C Quin, SD Vicaretti, NA Mohtarudin, AM Garner, DM Vollman, DL Gibson, WF Zandberg
J. Biol. Chem., 2020;0(0):.
Applications: Enzyme Assay
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Sulfotransferase Assays and Substrates
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