Recombinant Mouse CHST5 Protein, CF

Catalog # Availability Size / Price Qty
5210-ST-020
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Recombinant Mouse CHST5 Protein, CF Summary

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 >300 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived mouse Carbohydrate Sulfotransferase 5/CHST5 protein
Ser27-Ser395, with an N-terminal 6-His tag
Accession # NP_064334
Accession #
N-terminal Sequence
Analysis
His
Predicted Molecular Mass
42 kDa
SDS-PAGE
57 kDa, reducing conditions

Product Datasheets

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.

5210-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: 50 mM Tris, 15 mM MgCl2, pH 7.5
  • Recombinant Mouse Carbohydrate Sulfotransferase 5/CHST5 (rmCHST5) (Catalog # 5210-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
  1. 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.
  2. 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.
  3. Prepare reaction mixture containing 0.4 mM PAPS, 600 mM GlcNAc, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
  4. Dilute rmCHST5 to 4 µg/mL in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of the 4 µg/mL rmCHST5 into the plate. Include a Control containing 25 µL of Assay Buffer.
  7. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  8. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
  9. Add 30 µL of the Malachite Green Reagent A to all wells.  Mix briefly.
  10. Add 100 µL of deionized water to all wells. Mix briefly.
  11. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.
  12. Read plate at 620 nm (absorbance) in endpoint mode.
  13. 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:
  • rmCHST5: 0.1 μg
  • Coupling Phosphatase 3: 0.5 μg
  • PAPS: 0.2 mM
  • GlcNAc: 300 mM
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Carbohydrate Sulfotransferase 5/CHST5

The CHST family is comprised of 14 genes in both the human and mouse genomes. 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). CHST5, also designated as I-GlcNAc6ST, GlcNAc6ST-3, and GST-4 alpha (3, 4), has a marked preference for sulfating O-linked sugars of mucin type of acceptors (3) and catalyzes the transfer of sulfate to position 6 of non-reducing GlcNAc residues on keratan sulfate (5, 6). The amino acid sequence for mouse CHST5 has higher identity to that of human CHST6 (83%) than to human CHST5 (76%). Since there is no CHST6 found in the mouse genome it is possible that mouse CHST5 plays a similar biological role to the human CHST6 (6).The activity of the recombinant CHST6 has been measured with a phosphatase-coupled sulfotransferase assay (7).

References
  1. Hemmerich, S. and Rosen, S. (2000) Glycobiology 10:849.
  2. Bowman, K. G. and C. R. Bertozzi (1999) Chem. Biol. 5:447.
  3. Lee, J. K. et al. (2002) Glycobiology 13:245.
  4. Lee, J. K. et al. (1999) Biochem. Biophys. Res. Commun. 263: 543.
  5. Akama, T. O. et al. (2001) J. Biol. Chem. 276:16271.
  6. Akama, T. O. et al. (2002) J. Biol. Chem. 277:42505.
  7. Prather, B. et al. (2012) Anal. Biochem. 423:86.
Entrez Gene IDs
23563 (Human); 56773 (Mouse); 307859 (Rat)
Alternate Names
carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5; Carbohydrate Sulfotransferase 5; CHST5; GlcNAc6ST3; gn6st-3; GST4-alpha; hIGn6ST; I-GlcNAc6ST

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