Recombinant Human C1GalT1/C1GalT1C1 Protein, CF Summary
Product Specifications
Asp45-Pro363, with N-terminal 6-His tag (C1GalT1); Ile28-Asp318, with an N-terminal HA tag (C1GalT1C1)
Analysis
38 kDa (C1GalT1) & 35 kDa (C1GalT1C1)
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.
8659-GT
| 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 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
- 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.
- 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 1.2 mM UDP-Gal, 0.6 mM 4-NP-GalNAc, and 4 µg/mL Coupling Phosphatase 1 in Assay Buffer.
- Dilute rhC1GALT1 to 0.6 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 0.6 µg/mL rhC1GALT1 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 Well:
- rhC1GALT1: 0.015 µg
- Coupling Phosphatase 1: 0.1 µg
- UDP-Gal: 0.6 mM
- 4-NP-GalNAc: 0.3 mM
Reconstitution Calculator
Background: C1GalT1
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).
- Gerken, T.A. et al. (2011) J. Biol. Chem. 286:14493.
- Bergstrom, K.S.B. and Xia, L. (2013) Glycobiology 23:1026.
- Brockhausen, I. et al. (1999) Biochim. Biophys. Acta. 1473:67.
- Ju, T. and Cummings, R.D. (2005) Nature 437:1252.
- Fukuda, M. et al. (2002) Biochim. Biophys. Acta. 1573:394.
- Aryal, R.P. et al. (2012) J. Biol. Chem. 287:15317.
- Pirulli, D. et al. (2009) J. Nephrol. 22:152.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citation for Recombinant Human C1GalT1/C1GalT1C1 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.
1 Citation: Showing 1 - 1
-
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
FAQs
No product specific FAQs exist for this product, however you may
View all Proteins and Enzyme FAQsReviews for Recombinant Human C1GalT1/C1GalT1C1 Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Human C1GalT1/C1GalT1C1 Protein, CF and earn rewards!
Have you used Recombinant Human C1GalT1/C1GalT1C1 Protein, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
