Recombinant Human N-Acetylglucosaminyltransferase V/MGAT5 CF
Recombinant Human N-Acetylglucosaminyltransferase V/MGAT5 CF Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Leu189-Leu741, with a C-terminal 10-His tag
Analysis
Product Datasheets
5469-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.
5469-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and EDTA. |
| 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
- Buffer A: 25 mM MES, pH 6.0
- Buffer B: 100 mM Tris, 5 mM CaCl2, pH 7.5
- Recombinant Human N‑Acetylglucosaminyltransferase V/MGAT5 (rhMGAT5) (Catalog # 5469-GT)
- Donor Substrate: UDP-GlcNAc (Sigma, Catalog # U4375), 50 mM stock in 50% ethanol
- Acceptor Substrate: Biantennary N-Linked Core Pentasaccharide (V-Labs, Catalog # M592), 10 mM 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 Donor Substrate to 380 μM in Buffer A.
- Dilute Acceptor Substrate to 4 mM in Buffer A.
- Prepare Substrate Mixture by combining equal volumes of 380 μM Donor Substrate and 4 mM Acceptor Substrate.
- Dilute rhMGAT5 to 40 µg/mL in Assay Buffer.
- Dilute 1 mM Phosphate Standard by adding 40 µL of the Phosphate Standard to 360 µL of deionized water for a 100 µM stock.
- Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Buffer A. The standard curve has a range of 0.047 to 3 nmol per well.
- Load 60 µL of each dilution of the standard curve into a plate. Include a curve blank containing 60 μL of Buffer A.
- Load 20 µL of the 40 µg/mL rhMGAT5 into the plate. Include a substrate blank containing 20 µL of Buffer A.
- Start the primary reaction by adding 20 µL of Substrate Mixture to the wells, excluding the standard curve.
- Cover the plate with parafilm or a plate sealer and incubate at 37 ºC for 1 hour.
- Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
- Add 20 µL of 2 µg/mL Coupling Phosphatase 1 to reaction wells and blanks, excluding the standard curve (this is the secondary or coupling reaction).
- Mix and incubate for 10 minutes at room temperature.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 90 µL deionized water to all wells.
- 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 Substrate Blank.
**Based upon a 60 minute incubation time.
Per Reaction:- rhMGAT5: 0.8 µg
- Coupling Phosphatase 1: 40 ng
- Donor Substrate: 95 µM
- Acceptor Substrate: 1 mM
Background: N-Acetylglucosaminyltransferase V/MGAT5
N‑Acetylglucosaminyltransferase V (GnT‑V), also known as mannosylglycoprotein N‑acetyl-glucosaminyltransferase 5 (MGAT5), adds an N‑acetylglucosamine to the alpha 1‑6‑linked core mannose of an N‑linked oligosaccharide in the Golgi apparatus (1). This reaction is the committing step for the biosynthesis of beta 1‑6GlcNAc-branched arm in N‑glycans. The degree of N‑glycan branching has been shown to regulate cell proliferation and differentiation (2). An increase in the GnT-V activity and its glycan products is also known to positively correlate with the progression of invasive malignancies (3, 4). For example, ectopic expression of GnT‑V in epithelial cells results in morphological transformation and tumor growth in mice and overexpression in carcinoma cells has been shown to induce metastatic spread (3‑5).The enzymatic activity of recombinant human MGAT5 was determined using a phosphatase-coupled glycosyltransferase assay (6).
- Saito, H. et al. (1994) Biochem. Biophys. Res. Commun. 198:318.
- Lau, K.S. et al. (2007) Cell 198:123.
- Dennis, J.W. et al. (2002) Biochim. Biophys. Acta 1573:414.
- Granovsky, M. et al. (2008) Nat. Med. 7:1.
- Kim, Y.S. et al. (2008) Mol. Cell. Proteomics 7:1.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
Citation for Recombinant Human N-Acetylglucosaminyltransferase V/MGAT5 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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Phostine PST3.1a Targets MGAT5 and Inhibits Glioblastoma Initiating Cell Invasiveness and Proliferation
Authors: Z Hassani, A Saleh, S Turpault, S Khiati, W Morelle, J Vignon, JP Hugnot, E Uro-Coste, P Legrand, M Delaforge, S Loiseau, L Clarion, M Lecouvey, JN Volle, D Virieux, JL Pirat, H Duffau, N Bakalara
Mol. Cancer Res., 2017-06-20;0(0):.
Species: Human
Sample Types: Protein
Applications: Enzyme Assay
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