Recombinant Human GALNT12 Protein, CF
Recombinant Human GALNT12 Protein, CF Summary
Arg38-Leu581 with a C-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.
- Glycosyltransferase Activity Kit (Catalog # EA001)
- Assay Buffer: 25 mM Tris, 5 mM MnCl2, pH 7.5
- AssayRecombinant Human Polypeptide GalNac Transferase 12/GALNT12 (rhGALNT12) (Catalog # 9074-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
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Glycosyltransferase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
- Complete the standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare reaction mixture containing 2 mM UDP-GalNAc, 1 mM EA2 peptide, and 8 µg/mL Coupling Phosphatase 1 (supplied in kit) in Assay Buffer.
- Dilute rhGALNT12 to 10 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 10 µg/mL rhGALNT12 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
- Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
- Seal plate 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 sealed plate 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:
- rhGALNT12: 0.25 µg
- Coupling Phosphatase 1: 0.2 µg
- UDP- GalNac: 1 mM
- EA2 peptide: 0.5 mM
Background: Polypeptide GalNAc Transferase 12/GALNT12
O-glycosylation is a ubiquitous post-translational modification present in secreted and membrane-bound proteins. Polypeptide N-acetylgalactosaminyltransferases (GALNTs) catalyze 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). GALNT12 has activity toward non-glycosylated mucin peptides such as Muc5AC, Muc1a and EA2, and displays enzymatic activity toward mono-GalNAc-glycosylated Muc5AC glycopeptide, but no detectable activity to mono-GalNAc-glycosylated Muc1a, Muc2, Muc7 and EA2. It may play an important role in the initial step of mucin-type oligosaccharide biosynthesis in digestive organs (7). The enzymatic activity of recombinant human GALNT12 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.
- Guo, J.M. et al. (2002) FEBS Lett. 524:211.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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