Recombinant Human C1GalT1/C1GalT1C1 Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Human C1GalT1/C1GalT1C1 Protein, CF Summary

Details of Functionality
Measured by its ability to transfer galactose from UDP-galactose to 4-nitrophenyl-alpha -D-galactosaminide. The specific activity is >2,750 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human C1GalT1 protein
Asp45-Pro363, with N-terminal 6-His tag (C1GalT1); Ile28-Asp318, with an N-terminal HA tag (C1GalT1C1)
Accession #
N-terminal Sequence
His (C1GalT1) & Tyr (C1GalT1C1)
Protein/Peptide Type
Recombinant Proteins
Gene
C1GALT1
Purity
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW

38 kDa (C1GalT1) & 35 kDa (C1GalT1C1)

.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
25-42 kDa, reducing conditions
Publications
Read Publication using
8659-GT in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Purity
>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Assay Procedure
  • Glycosyltransferase Activity Kit (Catalog # EA001)
  • Assay Buffer: 25 mM Tris, 5 mM CaCl2, 10 mM MnCl2, pH 7.5
  • Recombinant Human C1GalT1 (rhC1GalT1) (Catalog # 8659-GT)
  • Uridine 5'-diphosphogalactose (UDP-Gal) (Sigma, Catalog # U4500), 10 mM stock in deionized water
  • 4-Nitrophenyl N-acetyl-alpha -D-galactosaminide (4-NP-GalNAc) (Sigma, Catalog # N4264), 15 mM stock in DMSO
  • 96-well Clear Plate (Catalog # DY990)
  • 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. This is the first point of the standard curve.
  2. 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.
  3. Prepare reaction mixture containing 1.2 mM UDP-Gal, 0.6 mM 4-NP-GalNAc, and 4 µg/mL Coupling Phosphatase 1 in Assay Buffer.
  4. Dilute rhC1GALT1 to 0.6 µg/mL 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 0.6 µg/mL rhC1GALT1 into empty wells of the same plate as the curve. Include a Control containing 25 μL of Assay Buffer.
  7. Add 25 µL of the reaction mixture to all wells, excluding the standard curve.
  8. Seal plate 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 sealed plate 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 Well:
  • rhC1GALT1: 0.015 µg
  • Coupling Phosphatase 1: 0.1 µg
  • UDP-Gal: 0.6 mM
  • 4-NP-GalNAc: 0.3 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human C1GalT1/C1GalT1C1 Protein, CF

  • B3Gal-T8
  • Beta-1,3-galactosyltransferase
  • C1GALT
  • C1GalT1
  • Core 1 beta1,3-galactosyltransferase 1
  • Core 1 Beta3-Gal-T1
  • Core 1 O-Glycan T-Synthase
  • core 1 synthase, glycoprotein-N-acetylgalactosamine3-beta-galactosyltransferase, 1
  • Core 1 UDP-galactose:N-acetylgalactosamine-alpha-R beta1,3-galactosyltransferase 1
  • EC 2.4.1.122
  • glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase 1
  • T-synthase

Background

O-glycosylation is a ubiquitous post-translational modification of secreted and membrane bound proteins (1). The synthesis of mucin-type O-glycans is initiated by the addition of GalNAc to threonine or serine residues on proteins by polypeptide N-acetylgalactosaminyltransferases (GALNTs) (2). The GalNAc alpha 1-O-Ser/Thr structure is then extended by other glycosyltransferases to form eight types of core O-glycans (3). Core 1 beta -3-galactosyltransferase (C1GalT1), in particular, synthesizes Gal-beta 1-3GalNAc alpha 1-O-Ser/Thr, a precursor for all core 1 and core 2 based mucin-type O-glycans (4). These glycans play central roles in many processes, such as angiogenesis, thrombopoiesis, and kidney homeostasis (5). C1GalT1 forms a stable, non-covalent complex with Cosmc chaperone, C1GalT1C1, which is required for the full activity of C1GalT1 (6). Defective C1GalT1 causes a rare autoimmune disease called Tn syndrome (4) as well as susceptibility to IgA nephropathy (7). The recombinant C1GalT1 is co-purified with C1GalT1C1. The enzymatic activity of recombinant human C1GalT1 was determined using a phosphatase-coupled assay (8).
  1. Gerken, T.A. et al. (2011) J. Biol. Chem. 286:14493.
  2. Bergstrom, K.S.B. and Xia, L. (2013) Glycobiology 23:1026.
  3. Brockhausen, I. et al. (1999) Biochim. Biophys. Acta. 1473:67.
  4. Ju, T. and Cummings, R.D. (2005) Nature 437:1252.
  5. Fukuda, M. et al. (2002) Biochim. Biophys. Acta. 1573:394.
  6. Aryal, R.P. et al. (2012) J. Biol. Chem. 287:15317.
  7. Pirulli, D. et al. (2009) J. Nephrol. 22:152.
  8. Wu, Z.L. et al. (2011) Glycobiology 21:727.

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Publications for C1GALT1 (8659-GT)(1)

We have publications tested in 1 confirmed species: Human.

We have publications tested in 1 application: Click Chemistry.


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