Recombinant Mouse CHST7 Protein, CF

Catalog #: 5108-ST Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
5108-ST-020

R&D Systems Recombinant Proteins and Enzymes

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All Carbohydrate Sulfotransferase 7/CHST7 Proteins and Enzymes

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Recombinant Mouse CHST7 Protein, CF Summary

Product Specifications

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.

Activity

Measured by its ability to transfer sulfate from PAPS to N-acetyl-D-glucosamine. The specific activity is >25 pmol/min/μg, as measured under the described conditions.

Source

Chinese Hamster Ovary cell line, CHO-derived mouse Carbohydrate Sulfotransferase 7/CHST7 protein
Ser34-Val484, with an N-terminal 6-His tag
Accession # Q9EP78

Accession #

Q9EP78

N-terminal Sequence
Analysis

His

Predicted Molecular Mass

52 kDa

SDS-PAGE

55-60 kDa, reducing conditions

Product Datasheets

5108-ST

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

5108-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.

Assay Procedure

Materials

Assay Buffer (provided in kit): 50 mM Tris, 15 mM MgCl₂, pH 7.5
Recombinant Mouse Carbohydrate Sulfotransferase 7/CHST7 (rmCHST7) (Catalog # 5108‑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 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, 30 mM GlcNAc, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
Dilute rmCHST7 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 rmCHST7 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:

rmCHST7: 0.25 μg
Coupling phosphatase 3: 0.5 μg
PAPS: 0.2 mM
GlcNAc: 15 mM

Reconstitution Calculator

Background: Carbohydrate Sulfotransferase 7/CHST7

The CHST family is comprised of 14 genes in both human and mouse. All members of this family are Golgi‑localized type II membrane proteins. 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 (1). This sulfation often creates specific epitopes that can be recognized by extracellular matrix proteins, cell surface receptors and viruses (2). CHST7, also designated as chondroitin 6‑O‑sulfotransferase (C6ST‑2), N‑acetylglucosamine-6‑O‑sulfotransferase (GlcNAc6ST-4) and Gal/GalNAc/GlcNAc 6‑O‑sulfotransferase (GST‑5), was previously shown to act on chondroitin sulfate, mannose‑linked GlcNAc and mucin associated GlcNAc residues (3‑5). The amino acid sequence of mouse CHST7 is 87% identical to that of the human counterpart. The enzymatic activity of the recombinant mouse CHST7 was measured using a phosphatase-coupled assay (6).

References