Recombinant Human GALNT3 Protein, CF Summary
Gln38-Asp633, with N-terminal 6-His tag
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: 25 mM Tris, 0.15 M NaCl, 5 mM MnCl2 (supplied in kit), 2.5 mM CaCl2, pH 7.5
- Recombinant Human Polypeptide GalNac Transferase 3/GALNT3 (rhGALNT3) (Catalog # 7174-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 (R&D Systems, Catalog# EA001)
- 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 Glycosyltransferase 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.
- Dilute EA2 peptide to 2 mM in Assay Buffer.
- Dilute Coupling Phosphatase I to 60 ng/µL in Assay Buffer.
- Prepare reaction mixture by combining 35 μL of 10 mM UDP-GalNAc, 87.5 μL of 2 mM EA2 peptide, 46.7 μL of 60 ng/μL Coupling Phosphatase I, and 180.8 μL Assay Buffer (sufficient to test 13 wells).
- Dilute rhGALNT3 to 4 ng/µL 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 ng/µL rhGALNT3 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 and curve blank.
- Cover the plate with parafilm or 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:
- rhGALNT3: 0.1 µg
- Coupling Phosphatase I: 0.2 µg
- EA2 peptide: 0.25 mM
- UDP-GalNAc: 0.5 mM
Background: Polypeptide GalNAc Transferase 3/GALNT3
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). Expression of GALNT3 appears to be highly regulated and mainly found in pancreas and testis (7). Using a peptide library screening approach, GALNT3 was classified as an intermediate transferase that increases the density of O-linked glycans within the mucin domain following glycosylation with early transferases (5). The enzymatic activity of recombinant human GALNT3 was determined using a phosphatase‑coupled assay (8).
- Gerken, T.A. et al. (2011) J. Biol. Chem. 286:14493.
- Ten Hagen, K.G. et al. (2003) Glycobiology 13:1R.
- Hagen, F.K. et al. (1997) J. Biol. Chem. 272:13843.
- Gerken, T.A. et al. (2006) J. Biol. Chem. 281:32403.
- Wandall, H.H. et al. (1997) J. Biol. Chem. 272:23503.
- Pratt, M.R. et al. (2004) Chem. Biol. 11:1009.
- Bennett, E.P. et al. (1996) J. Biol. Chem. 271:17006.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
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Glycosyltransferase Activity Assays
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