>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
<1.0 EU per 1 μg of the protein by the LAL method.
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.
25-42 kDa, reducing conditions
Read Publication using 8659-GT in the following applications:
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
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
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.
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PRODUCT AVAILABILITY: Update Regarding the Evolving COVID-19 Situation
Bio-Techne appreciates the critical role that you and our products and services play in research efforts to further scientific innovation and discovery. We are continually assessing our manufacturing and supplier capabilities during the COVID-19 situation and are implementing precautionary measures to ensure uninterrupted supply of products and services. Currently, and as we abide by local shelter in place orders across the world, we are fully operational and do not anticipate any material supply disruptions across our Bio-Techne brands and product lines. As the situation evolves, our goal is to utilize preventive measures to reduce the threat that COVID-19 poses to our ability to meet the needs of our customers globally.