Recombinant Human CHST15 Protein, CF

R&D Systems | Catalog # 3365-ST

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human CHST15 Protein (3365-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

NS0

Accession Number

Q7LFX5

Applications

Enzyme Activity
View all Carbohydrate Sulfotransferase 15/CHST15 Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human Carbohydrate Sulfotransferase 15/CHST15 protein
Ser99-Thr561, with an N-terminal 6-His tag

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

55 kDa

SDS-PAGE

67 kDa and 72 kDa, reducing conditions

Activity

Measured by its ability to transfer sulfate from PAPS to chondroitin sulfate.
The specific activity is >100 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

3365-ST
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: Carbohydrate Sulfotransferase 15/CHST15

N‑Acetylgalactosamine 4‑sulfate 6‑O‑sulfotransferase (GalNAc4S‑6ST, or, CHST15) is a type II transmembrane protein composed of a short N‑terminal cytoplasmic domain and a long C‑terminal luminal catalytic domain. GalNAc4S‑6ST transfers sulfate from 3'‑phosphoadenosine 5'‑phosphosulfate (PAPS) to the 6‑O of GalNAc‑4S on chondroitin sulfate A (CS‑A), as well as dermatan sulfate (1), generating the chondroitin sulfate E (CS‑E) disaccharide unit GlcA beta 1‑3GalNAc‑4S,6S. CS‑E binds with strong affinity to Midkine, a heparan sulfate‑binding growth factor (2), playing an important regulatory role in differentiation and morphogenesis during embryonic development. In situ hybridization for the expression of GalNAc4S‑6ST in the postnatal mouse brain showed a widespread expression of the transcript in the developing brain except at postnatal day 7 (3). For comparison, chondroitin sulfate 6‑O sulfotransferase‑1 (C6ST‑1) encoded by the CHST3 gene, only recognizes and transfers sulfate to the 6‑O of unsulfated GalNAc residues on chondroitin sulfate generating the chondroitin sulfate C (CS‑C) disaccharide unit GlcA beta 1‑3GalNAc‑6S (4). The sequence of the human GalNAc4S‑6ST was found to be the same as human B‑cell RAG‑associated protein (BRAG) (1). The activity of the recombinant human CHST15 is measured using a PAP-specific phosphatasecoupled sulfotransferase assay (5).

References

  1. Ohtake, S. et al. (2001) J. Biol. Chem. 276:43894.
  2. Zou, P. et al. (2003) Glycobiology 13:35.
  3. Purushothaman, A. et al. (2007) J. Biol. Chem. 282:19442.
  4. Thiele, H. et al. (2004) Proc. Natl. Acad. Sci. USA 101:10155.
  5. Prather, B. et al. (2012) Anal. Biochem. 423:86.

Alternate Names

BRAG, CHST15, GalNAc4S-6ST

Entrez Gene IDs

51363 (Human); 286974 (Rat)

Gene Symbol

CHST15

UniProt

Q7LFX5

Additional Carbohydrate Sulfotransferase 15/CHST15 Products

Product Documents for Recombinant Human CHST15 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human CHST15 Protein, CF

For research use only

Citations for Recombinant Human CHST15 Protein, CF

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Protocols

View specific protocols for Recombinant Human CHST15 Protein, CF (3365-ST):

Materials
  • Universal Sulfotransferase Activity Kit (Catalog # EA003)
  • 10X Assay Buffer (supplied in kit): 500 mM Tris, 150 mM MgCl2, pH 7.5
  • Recombinant Human Carbohydrate Sulfotransferase 15/CHST15 (rhCHST15) (Catalog # 3365-ST)
  • 3'-Phosphoadenosine-5'-Phosphosulfate (PAPS) (Catalog # ES019)
  • Chondroitin Sulfate (Sigma, Catalog # C6737), 50 mg/mL stock in deionized water
  • 96-well Clear Plate (Catalog # DY990)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Prepare 1X Assay Buffer by diluting 10X stock 10 fold with deionized water.
  2. Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of 1X Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
  3. Continue standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
  4. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
  5. Prepare a reaction mixture containing 0.4 mM PAPS, 10 mg/mL Chondroitin Sulfate, and 20 µg/mL Coupling Phosphatase 3 in 1X Assay Buffer.
  6. Dilute rhCHST15 to 20 µg/mL in 1X Assay Buffer.
  7. Load 25 µL of the 20 µg/mL rhCHST15 into empty wells of the same plate as the curve. Include a control containing 25 µL of Assay Buffer.
  8. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  9. Seal plate and incubate at room temperature for 20 minutes.
  10. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
  11. Add 100 µL of deionized water to all wells. Mix briefly.
  12. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  13. Read plate at 620 nm (absorbance) in endpoint mode.
  14. 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 fitting and adjusted for Control.

Per Reaction:

  • rhCHST15: 0.5 μg
  • Coupling Phosphatase 3: 0.5 μg
  • Chondroitin Sulfate: 250 μg
  • PAPS:  0.2 mM

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