Recombinant Human CHST4 Protein, CF
Recombinant Human CHST4 Protein, CF Summary
Product Specifications
His29-His386, with an N-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
5357-ST
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| 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 Procedure
- Assay Buffer (1X Phosphatase Buffer 3): 50 mM Tris, 15 mM MgCl2, pH 7.5
- Recombinant Human Carbohydrate Sulfotransferase 4/CHST4 (rhCHST4) (Catalog # 5357-ST)
- 3'-Phosphoadenosine-5'-phosphosulfate (PAPS) (Catalog # ES019)
- N-acetyl-alpha -D-glucosamine (GlcNAc) (Calbiochem, Catalog # 1079), 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 by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
- Continue 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 5.0 nmol per well.
- Prepare a reaction mixture containing 0.4 mM PAPS, 20 mM GlcNAc and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
- Dilute rhCHST4 to 10 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 10 µg/mL rhCHST4 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:- rhCHST4: 0.25 µg
- Coupling Phosphatase 3: 0.500 μg
- PAPS: 0.2 mM
- GlcNAc: 10 mM
Reconstitution Calculator
Background: Carbohydrate Sulfotransferase 4/CHST4
The CHST family is comprised of 14 enzymes in human. All members of this family are Golgi-localized type II membrane proteins (1). Only the luminal and enzymatic domain is expressed in each of our recombinant CHST proteins. These enzymes transfer sulfate (i.e., sulfonate) onto the 6-O or 4-O positions of GalNAc, Gal and GlcNAc residues on glycoproteins, proteoglycans and glycolipids (2). This sulfation often creates specific epitopes that can be recognized by extracellular matrix proteins, cell surface receptors and viruses (3). CHST4, also known as high endothelial cells N-acetylglucosamine 6-O-sulfotransferase (HEC-GlcNAc6ST) or L‑selectin ligand sulfotransferases (LSST), catalyzes the transfer of sulfate to position 6 of non-reducing GlcNAc residues within mucin-associated glycans that ultimately serve as L‑selectin ligands (4). It has a catalytic preference for core 2-branched mucin-type O‑glycans, but also has activity toward core 3 type of O‑glycan (5). Human CHST4 shares 72% amino acid sequence identity with the mouse ortholog.The enzymatic activity of the recombinant human CHST4 is measured using a phosphatase-coupled assay (6).
- deGraffenried, D. and Bertozzi, C.R. (2003) J. Biol. Chem. 278:40282.
- Hemmerich, S. and Rosen, S. (2000) Glycobiology 10:849.
- Bowman, K. G. and Bertozzi, C. R. (1999) Chem. Biol. 5:447.
- Bistrup, A. et al. (1999) J. Cell Biol. 145:899.
- Uchimura, K. et al. (2002) J. Biol. Chem. 277:3979.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
Citations for Recombinant Human CHST4 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.
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Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases.
Authors: Wu ZL, Prather B, Ethen CM
Glycobiology, 2010-12-17;21(5):625-33.
Species: Bovine, Porcine
Sample Types: Heparin, Protein
Applications: Enzyme Assay -
A versatile polyacrylamide gel electrophoresis based sulfotransferase assay.
Authors: Wu ZL, Ethen CM, Larson S, Prather B, Jiang W
BMC Biotechnol., 2010-02-10;10(0):11.
Applications: Enzyme Assay
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