Recombinant Human GCNT1 Protein, CF
Recombinant Human GCNT1 Protein, CF Summary
Product Specifications
Arg33-His428, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
7248-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35. |
| 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 Procedure
- Assay Buffer: 0.1 M MES, 10 mM DTT, 5 mM CaCl2, pH 6.0
- Recombinant Human Glucosaminyl (N-acetyl) Transferase 1/GCNT1 (rhGCNT1) (Catalog # 7248-GT)
- UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
- beta 1-3 Galactosyl-N-acetyl galactosamine (V-labs, Catalog # GN213), 20 mM stock in deionized water
- Glycosyltransferase Activity Kit (Catalog # EA001)
- 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 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 a reaction mixture containing 0.4 mM UDP-Glc-NAc, 1 mM beta 1-3 Galactosyl-N-acetyl galactosamine and 4 μg/mL Coupling Phosphatase I in Assay Buffer.
- Dilute rhGCNT1 to 5 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 5 ng/µL rhGCNT1 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.
- Seal the 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 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:- rhGCNT1: 0.125 µg
- Coupling Phosphatase I: 0.1 µg
- beta 1-3 Galactosyl-N-acetyl galactosamine: 0.5 mM
- UDP-GlcNAc: 0.2 mM
Reconstitution Calculator
Background: Glucosaminyl (N-acetyl) Transferase 1/GCNT1
Mucin-type O-glycans are initiated with an O-GalNAc attachment to a serine or threonine in a polypeptide. The O-GalNAc residues are subsequently extended by various glycosyltransferases resulting in different types of O-glycans. Most O-glycans contain the core 1 structure, Gal beta 1-3GalNAc. Glucosaminyl (N-acetyl) Transferase 1 (GCNT1) converts the core 1 O-glycan to core 2 O-glycan, Gal beta 1-3(GlcNAc beta 1-6)GalNAc, via the addition of a GlcNAc residue (1, 2). Various ligand carbohydrates can be formed from core 2 branched oligosaccharides. For example, sialyl Lex in mucin‑type glycoproteins of blood cells can be formed from core 2 branched oligosaccharides (3, 4). The expression of GCNT1 was found to be associated with the progression of various types of cancer (5, 6, 7). The enzymatic activity of the recombinant GCNT1 is measured using a phosphatase-coupled method (8).
- Bierhuizen, M.F. (1993) Genes & Development 7:468.
- Yeh, J.C. et al. (1999) J. Biol. Chem. 274:3215.
- Hemmerich, S. et al. (1995) J. Biol. Chem. 270:12035.
- Wilkins, P.P. et al. (1996) J. Biol. Chem. 270:18732.
- Shimodaira, K. et al. (1997) Cancer Res. 57: 5201.
- Hatakyama, S. et al. (2010) Int. J. Cancer 127:1052.
- St Hill, C.A. et al. (2009) BMC Cancer 9:79.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citations for Recombinant Human GCNT1 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 4
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Fluorescent glycan fingerprinting of SARS2 spike proteins
Authors: ZL Wu, JM Ertelt
Scientific Reports, 2021-10-14;11(1):20428.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay -
Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA from Akkermansia muciniphila
Authors: B Trastoy, A Naegeli, I Anso, J Sjögren, ME Guerin
Nat Commun, 2020-09-24;11(1):4844.
Species: E. coli
Sample Types: Peptide
Applications: Bioassay -
Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.
Authors: Wu Z, Person A, Anderson M, Burroughs B, Tatge T, Khatri K, Zou Y, Wang L, Geders T, Zaia J, Sackstein R
Glycobiology, 2018-02-01;0(0):.
Species: Human, Mouse
Sample Types: Whole Cells
Applications: Click Chemistry -
Detection of Core2 beta-1,6-N-Acetylglucosaminyltransferase in Post-Digital Rectal Examination Urine Is a Reliable Indicator for Extracapsular Extension of Prostate Cancer.
Authors: Kojima Y, Yoneyama T, Hatakeyama S, Mikami J, Sato T, Mori K, Hashimoto Y, Koie T, Ohyama C, Fukuda M, Tobisawa Y
PLoS ONE, 2015-09-21;10(9):e0138520.
Species: Human
Sample Types: Urine
Applications: Western Blot
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