Recombinant Human GALNT4 Protein, CF

R&D Systems | Catalog # 7528-GT

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human GALNT4 Protein (7528-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

Q8N4A0

Applications

Enzyme Activity
View all Polypeptide GalNAc Transferase 4/GALNT4 Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human Polypeptide GalNac Transferase 4/GALNT4 protein
Ala36-Lys578, with C-terminal 6-His tag

Purity

>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Ala36

Predicted Molecular Mass

64 kDa

SDS-PAGE

57-62 kDa, reducing conditions

Activity

Measured by its ability to transfer GalNAc from UDP-GalNAc to peptide EA2 from AnaSpec, Inc.
The specific activity is >200 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

7528-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: Polypeptide GalNAc Transferase 4/GALNT4

O-glycosylation is a ubiquitous post-translational modification present in secreted and membrane-bound proteins. Polypeptide N-acetylgalactosaminyltransferases (GALNTs) calalyze the initial step for O-glycosylation by transferring GalNAc to Thr or Ser residues (GalNAc alpha 1-O-Ser/Thr) in the Golgi compartment. Structurally, the GALNTs consist of an N-terminal catalytic domain tethered by a short linker to a C-terminal ricin-like lectin domain containing three potential carbohydrate-binding sites (1, 2). Twenty distinct GALNT isoforms have been detected in humans. These isoforms display both unique and overlapping substrate specificities (3, 4, 5) with no known universal consensus glycosylation sequence. Glycosylation of mucins results from the successive, often hierarchical, action of several specific GALNTs (6). Human GALNT4 is widely expressed with particularly high expression in the immune system (7). The enzymatic specificity of GALNT4 depends on the selective binding between the C-terminal lectin domain and the existing O-GalNAc modified residues present within the substrate polypeptide (8). Along with GALNT3, GALNT4 is classified as an intermediate transferase that glycosylates mucin polypeptides that have been previously mono- or di-glycosylated by other transferases (6). The enzymatic activity of recombinant human GALNT4 was determined using a phosphatase-coupled assay (9).

References

  1. Gerken, T.A. et al. (2011) J. Biol. Chem. 286:14493.
  2. Ten Hagen, K.G. et al. (2003) Glycobiology 13:1R.
  3. Hagen, F.K. et al. (1997) J. Biol. Chem. 272:13843.
  4. Gerken, T.A. et al. (2006) J. Biol. Chem. 281:32403.
  5. Wandall, H.H. et al. (1997) J. Biol. Chem. 272:23503.
  6. Pratt, M.R. et al. (2004) Chem. Biol. 11:1009.
  7. Bennett, E.P. et al. (1998) J. Biol. Chem. 273:30472.
  8. Hassan, H. et al. (2000) J. Biol. Chem. 275:38197.
  9. Wu, Z.L. et al. (2011) Glycobiology 21:727.

Long Name

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 4

Alternate Names

GALNACT4

Entrez Gene IDs

8693 (Human); 14426 (Mouse); 500826 (Rat)

Gene Symbol

GALNT4

UniProt

Q8N4A0 (Human)

Additional Polypeptide GalNAc Transferase 4/GALNT4 Products

Product Documents for Recombinant Human GALNT4 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human GALNT4 Protein, CF

For research use only

Related Research Areas

Glycobiology-related Enzymes

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Protocols

View specific protocols for Recombinant Human GALNT4 Protein, CF (7528-GT):

Materials
  • Assay Buffer: 25 mM Tris, 5 mM MnCl2 (supplied in kit), 2.5 mM CaCl2, pH 7.5
  • Recombinant Human Polypeptide GalNac Transferase 4/GALNT4 (rhGALNT4) (Catalog # 7528-GT)
  • UDP-GalNAc (Sigma, Catalog # U5252), 10 mM stock in deionized water
  • EA2 peptide (AnaSpec Inc, Catalog # 63841), 5 mM in 5 mM Tris, pH 7.0
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • 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 Glycosyltransferase 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 a reaction mixture composed of 1 mM UDP-GalNAc, 0.5 mM EA2 peptide, and 4 ng/µL Coupling Phosphatase I in Assay Buffer.
  4. Dilute rhGALNT4 to 8 ng/µL 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 8 ng/µL rhGALNT4 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 and curve blank.
  8. Cover the plate with parafilm or 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:

  • rhGALNT4: 0.2 µg
  • Coupling Phosphatase I: 0.1 µg
  • EA2 peptide: 0.25 mM
  • UDP-GalNAc: 0.5 mM

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