Recombinant Human CHST15 Protein, CF Summary
Ser99-Thr561, with an N-terminal 6-His tag
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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.
- 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
- Prepare 1X Assay Buffer by diluting 10X stock 10 fold with deionized water.
- 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.
- 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.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- 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.
- Dilute rhCHST15 to 20 µg/mL in 1X Assay Buffer.
- 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.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Seal plate and incubate at room temperature 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 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
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).
- Ohtake, S. et al. (2001) J. Biol. Chem. 276:43894.
- Zou, P. et al. (2003) Glycobiology 13:35.
- Purushothaman, A. et al. (2007) J. Biol. Chem. 282:19442.
- Thiele, H. et al. (2004) Proc. Natl. Acad. Sci. USA 101:10155.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
Citation for Recombinant Human CHST15 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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