Recombinant Human ST6GALNAC4 Protein, CF

R&D Systems | Catalog # 6876-GT

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human ST6GALNAC4 Protein (6876-GT)
  • 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

Q9H4F1

Applications

Enzyme Activity
View all ST6GALNAC4 Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human ST6GALNAC4 protein
Thr36-Arg302, with a C-terminal 6-His tag

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Thr36 and Leu38

Predicted Molecular Mass

31 kDa

SDS-PAGE

36-38 kDa, reducing conditions

Activity

Measured by its ability to transfer Neu5Ac from CMP-Neu5Ac to fetuin of fetal calf serum.
The specific activity is >1000 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

6876-GT
Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Shipping The product is shipped with dry ice or equivalent. 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, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Background: ST6GALNAC4

ST6GALNAC4 is a type II membrane protein that catalyzes the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates (1). This enzyme works on both glycoproteins and glycolipids and has strict substrate specificity, utilizing only the trisaccharide sequence Neu5Ac alpha 2-3Gal beta 1-3GalNAc of glycoproteins and glycolipids (2). In particular, this enzyme is involved in the synthesis of ganglioside GD1A from GM1B (3). GD1A is the target pathogenic antigen in the autoimmune disease, Guillain-Barre Syndrome (4, 5). The activity of this enzyme has been measured using a phosphatase-coupled method (6).

References

  1. Harduin-Lepers, A. et al. (2005) Glycobiology 15:805.
  2. Harduin-Lepers, A. et al. (2000) Biochem. J. 352:37.
  3. Lee, Y. C. et al. (1999) J. Biol. Chem. 274:11958.
  4. Kusunoki, S. et al. (1994) Ann. Neurol. 35:570.
  5. Goodfellow, J.A. et al. (2005) J. Neurosci. 25:1620.
  6. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Alternate Names

SIAT3C, SIAT7D

Entrez Gene IDs

27090 (Human); 20448 (Mouse)

Gene Symbol

ST6GALNAC4

UniProt

Q9H4F1

Additional ST6GALNAC4 Products

Product Documents for Recombinant Human ST6GALNAC4 Protein, CF

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot or batch number in the search box below.

Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human ST6GALNAC4 Protein, CF

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

For research use only

Related Research Areas

Glycobiology-related Enzymes

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Protocols

View specific protocols for Recombinant Human ST6GALNAC4 Protein, CF (6876-GT):

Materials
  • Assay Buffer: 50 mM Tris, 10 mM MnCl2 (supplied in kit), pH 7.0
  • Recombinant Human ST6GALNAC4 (rhST6GALNAC4) (Catalog # 6876-GT)
  • Donor Substrate: CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
  • Acceptor Substrate: Fetuin (Sigma, Catalog # F3385), 50 mg/mL stock in diH20
  • Sialyltransferase Activity Kit (Catalog # EA002)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute CMP-Sialic Acid to 2.5 mM in Assay Buffer.
  2. Dilute Coupling Phosphatase 2 to 10 µg/mL in Assay Buffer.
  3. Prepare reaction mixture by combining 75 µL of 2.5 mM CMP-Sialic Acid, 150 µL of 10 µg/mL Coupling Phosphatase 2, 75 µL of 50 mg/mL Fetuin, and 75 µL of Assay Buffer.
  4. Dilute rhST6GALNAC4 to 0.8 µg/mL in Assay Buffer.
  5. Dilute 1 mM Phosphate Standard by adding 40 µL to 360 µL of Assay Buffer for a 100 μM stock. This is the first point of the standard curve.
  6. Continue 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 5.0 nmol per well.
  7. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
  8. Load 25 µL of the 0.8 µg/mL rhST6GALNAC4 into the plate. Include a control containing 25 µL of Assay Buffer.
  9. Add 25 µL of reaction mixture to the wells, excluding the standard curve.
  10. Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 20 minutes.
  11. Add 30 µL of the Malachite Green Reagent A to all wells.  Mix and incubate for 10 minutes at room temperature.
  12. Add 100 µL of deionized water to all wells. Mix briefly.
  13. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  14. Read plate at 620 nm (absorbance) in endpoint mode.
  15. 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 the control.

Per Reaction:

  • rhST6GALNAC4: 0.02 µg
  • Coupling Phosphatase 2: 0.1 µg
  • Fetuin: 250 µg
  • CMP-Sialic Acid: 0.25 mM

FAQs

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