Recombinant Human POMGNT1 Protein, CF Summary
Product Specifications
Ser66-Thr660, with a C-terminal 6-His tag
Analysis
Product Datasheets
7414-GT
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.
7414-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| 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: 25 mM MES, 5 mM MnCl2, 2.5 mM CaCl2, 0.02% Brij-35, pH 6.5
- Recombinant Human POMGNT1 (rhPOMGNT1) (Catalog # 7414-GT)
- UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
- Methyl-alpha -D-mannopyranoside (Sigma, Catalog # M6882), 1 M stock in deionized water
- Glycosyltransferase Activity Kit (Catalog # EA001)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided in the 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 seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
- Dilute Coupling Phosphatase I to 24 µg/mL in Assay Buffer.
- Dilute UDP-GlcNAc to 6 mM in Assay Buffer.
- Prepare Reaction Mixture by combining equal volumes of 24 µg/mL Coupling Phosphatase I, 6 mM UDP-GlcNAc, and 1 M methyl‑ alpha ‑D‑mannopyranoside.
- Dilute rhPOMGNT1 to 2 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 the 2 µg/mL rhPOMGNT1 into the plate. Include a Control containing 25 µL of Assay Buffer.
Start the reaction by adding 25 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank. - Cover the plate with a plate sealer and incubate at 37 ºC 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:Background: POMGNT1
Protein O-mannose beta -1,2-N-acetylglucosaminyltransferase (POMGNT1) is a Golgi-resident type II transmembrane protein that participates in O-mannosyl glycosylation (1, 2). In particular, POMGNT1 is involved in the synthesis of the GlcNAc beta 1-2Man alpha 1-O-Ser/Thr moiety present on alpha -dystroglycan and other O‑mannosylated proteins (3). A phosphorylated O‑mannosyl glycan is identified as a laminin-binding ligand of alpha -dystroglycan (4) that functions as part of the transmembrane linkage between the extracellular matrix and the cytoskeleton in skeletal muscle. POMGNT1 is widely expressed in all tissues, with the highest levels found in skeletal muscle and the heart (1, 2). Mutations of POMGNT1 are associated with muscle-eye-brain (MEB) disease (5, 6, 7). MEB is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, cobblestone lissencephaly and cerebellar hypoplasia. The enzymatic activity of the recombinant human POMGNT1 was determined using methyl-alpha -D-mannopyranoside as the acceptor substrate with a phosphatase-coupled glycosyltransferase assay (7).
- Zhang, W. et al. (2002) Biochem. J. 361:153.
- Yoshida, A. et al. (2001) Dev. Cell 1:717.
- Chiba, A. et al. (1997) J. Biol. Chem. 272:2156.
- Yoshida-Moriguchi, T. et al. (2010) Science 327:88.
- Manya, H. et al. (2003) Bichem. Biophys. Res. Commun. 320:39.
- Taniguchi, K. et al. (2003) Hum. Mol. Genet. 12:527.
- Diesen, C. et al. (2004) J. Med. Genet. 41:e115.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.Citation for Recombinant Human POMGNT1 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
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Homozygosity Mapping and Whole-Genome Sequencing Links a Missense Mutation in POMGNT1 to Autosomal Recessive Retinitis Pigmentosa
Invest Ophthalmol Vis Sci, 2016-07-01;57(8):3601-9.
Species: Human
Sample Types: Protein
Applications: Bioassay
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