Recombinant Mouse CHST5 Protein, CF Summary
Ser27-Ser395, with an N-terminal 6-His tag
Accession # NP_064334
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.
- 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
- 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, 600 mM GlcNAc, and 20 μg/mL Coupling Phosphatase 3 in Assay Buffer.
- Dilute rmCHST5 to 4 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 4 µg/mL rmCHST5 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:
- rmCHST5: 0.1 μg
- Coupling Phosphatase 3: 0.5 μg
- PAPS: 0.2 mM
- GlcNAc: 300 mM
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).
- Hemmerich, S. and Rosen, S. (2000) Glycobiology 10:849.
- Bowman, K. G. and C. R. Bertozzi (1999) Chem. Biol. 5:447.
- Lee, J. K. et al. (2002) Glycobiology 13:245.
- Lee, J. K. et al. (1999) Biochem. Biophys. Res. Commun. 263: 543.
- Akama, T. O. et al. (2001) J. Biol. Chem. 276:16271.
- Akama, T. O. et al. (2002) J. Biol. Chem. 277:42505.
- Prather, B. et al. (2012) Anal. Biochem. 423:86.
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Sulfotransferase Assays and Substrates
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