The CHST family is comprised of 14 genes in humans. All members of this family are Golgi-localized type II membrane proteins. Only the luminal and enzymatic domain is expressed in each of R&D Systems 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 (1). This sulfation often creates specific epitopes that can be recognized by extracellular matrix proteins, cell surface receptors and viruses (2). CHST6, also designated as corneal N-acetylglucosamine-6-O-sulfotransferase (3) or N-acetylglucosamine 6‑O‑sulfotransferase 5 (1), transfers sulfate to position 6 of non‑reducing GlcNAc residues of keratan sulfate found in the corneal stroma. It also acts on the non‑reducing terminal GlcNAc residues of carbohydrate substrates that have poly-N-acetyllactosamine structures (1). Defects in CHST6 cause macular corneal dystrophy, an autosomal recessive disease characterized by corneal opacities (3). The activity of the recombinant CHST6 has been measured with a phosphatase-coupled sulfotransferase assay (4).
Recombinant Human CHST6 Protein, CF
R&D Systems | Catalog # 5326-ST
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Key Product Details
- R&D Systems CHO-derived Recombinant Human CHST6 Protein (5326-ST)
- Quality control testing to verify active proteins with lot specific assays by in-house scientists
- All R&D Systems proteins are covered with a 100% guarantee
Source
CHO
Accession Number
Applications
Enzyme Activity
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Product Specifications
Source
Chinese Hamster Ovary cell line, CHO-derived human Carbohydrate Sulfotransferase 6/CHST6 protein
Ser27-Asn395, with an N-terminal 6-His tag
Accession # NP_067628
Ser27-Asn395, with an N-terminal 6-His tag
Accession # NP_067628
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
His
Predicted Molecular Mass
42 kDa
SDS-PAGE
55 kDa, under reducing conditions
Activity
Measured by its ability to transfer sulfate from PAPS to N-acetyl-D-glucosamine.
The specific activity is >50 pmol/min/μg, as measured under the described conditions.
The specific activity is >50 pmol/min/μg, as measured under the described conditions.
Formulation, Preparation, and Storage
5326-ST
| 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.
|
Background: Carbohydrate Sulfotransferase 6/CHST6
References
- Hemmerich, S. and Rosen, S. (2000) Glycobiology 10:849.
- Bowman, K. G. and Bertozzi, C. R. (1999) Chem. Biol. 5:447.
- Akama, T. O. et al. (2000) Nat. Genet. 26:237.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
Alternate Names
C-GlcNAc6ST, CGn6ST, CHST6, GlcNAc6ST-5, GST4-beta, MCDC1
Gene Symbol
CHST6
UniProt
Additional Carbohydrate Sulfotransferase 6/CHST6 Products
Product Documents for Recombinant Human CHST6 Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Human CHST6 Protein, CF
For research use only
Related Research Areas
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Protocols
View specific protocols for Recombinant Human CHST6 Protein, CF (5326-ST):
Materials
- Assay Buffer: 50 mM Tris, 15 mM MgCl2, pH 7.5
- Recombinant Human Carbohydrate Sulfotransferase 6/CHST6 (rhCHST6) (Catalog # 5326-ST)
- 3'-Phosphoadenosine-5'-phosphosulfate/PAPS (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 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, 200 mM GlcNAc, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
- Dilute rhCHST6 to 40 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 40 µg/mL rhCHST6 into the plate. Include a Substrate Blank 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:
- rhCHST6: 1.0 μg
- Coupling Phosphatase 3: 0.5 μg
- PAPS: 0.2 mM
- GlcNAc: 100 mM
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Associated Pathways
