Recombinant Human CHST10 Protein, CF Summary
Pro32-Asn356, with a C-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 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 (provided in kit): 50 mM Tris, 15 mM MgCl2, pH 7.5
- Recombinant Human Carbohydrate Sulfotransferase 10/CHST10 (rhCHST10) (Catalog # 6140-ST)
- 3'-Phosphoadenosine-5'-phosphosulfate/PAPS (Catalog # ES019)
- Phenolphthalein glucuronic acid (PGA) (Sigma, Catalog # P0501), 0.1 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 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 0.4 mM PAPS, 1 mM PGA, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
- Dilute rhCHST10 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 rhCHST10 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. 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.Per Reaction:
- rhCHST10: 0.1 μg
- Coupling Phosphatase 3: 0.5 μg
- PAPS: 0.2 mM
- PGA: 0.5 mM
Background: Carbohydrate Sulfotransferase 10/CHST10
CHST10 is the only known sulfotransferase that catalyzes the transfer of sulfate to position 3 of terminal glucuronic acid of both protein- and lipid-linked oligosaccharides (1, 2). CHST10 is a key enzymes for the synthesis of HNK-1 carbohydrate structure, a sulfated glucuronyl-lactosaminyl residue carried by many neural recognition molecules (3). HNK-1 is involved in cell interactions during ontogenetic development and in synaptic plasticity in the adult. In adults, CHST10 is highly expressed in brain, testis and ovary. Recently, it is reported that CHST10 functions as a suppressor of invasiveness of human melanoma cells (4). Like many other carbohydrate specific sulfotransferases, CHST10 is a single-pass type II membrane protein and is localized in Golgi apparatus membrane. The recombinant human CHST10 only contains the luminal enzymatic domain and the enzymatic activity is measured using a phosphatase-coupled assay (5).
- Ong, E. et al. (1999) J. Biol. Chem. 274:25608.
- Ong, E. et al. (1998) J. Biol. Chem. 273:5190.
- Kizuka, Y. et al. (2006) J. Biol. Chem. 281:13644.
- Zhao, X. et al. (2009) Cancer Res. 69:5218.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
Citation for Recombinant Human CHST10 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
Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.
Authors: Wu ZL, Prather B, Ethen CM
Sample Types: Protein
Applications: Enzyme Assay
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Sulfotransferase Assays and Substrates
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