Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 CF Summary
Details of Functionality
Measured by its ability to transfer N-Acetyl-alpha -D-glucosamine from UDP-N-Acetyl-alpha -D-glucosamine to a biantennary N-linked core pentasaccharide in a CD39L3 coupled assay. The specific activity is >100 pmol/min/μg, as measured under the described conditions.
Chinese Hamster Ovary cell line, CHO-derived Tyr31-Val533, with a C-terminal 6-His tag
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
<1.0 EU per 1 μg of the protein by the LAL method.
58 kDa. 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.
42-54 & 64-70 kDa, reducing conditions
Read Publication using 7359-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 Buffer A for a 100 µM stock.
Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Buffer A. The standard curve has a range of 0.078 to 5 nmol per well.
Dilute rhMGAT3 to 10 µg/mL in Buffer A.
Dilute Donor Substrate to 1 mM in Buffer A.
Dilute Acceptor Substrate to 1 mM in Buffer A.
Prepare Substrate Mixture by combining 120 µL of 1 mM Donor Substrate, 120 µL of 1 mM Acceptor Substrate, and 60 µL of Buffer A.
Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Buffer A.
Load 25 µL of the 10 µg/mL rhMGAT3 into the plate. Include a Control containing 25 µL of Buffer A.
Start the reaction by adding 25 µL of Substrate Mixture to the wells, excluding the standard curve.
Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
Add 50 µL of 2 µg/mL Coupling Phosphatase 1 to reaction wells and blanks, excluding the standard curve. Also, add 50 µL of Buffer B to the wells containing the standard curve. Mix and incubate for 10 minutes at room temperature.
Add 30 µL of the Malachite Green Reagent A to all wells.
Add 50 µL of 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 Control.
rhMGAT-3: 0.25 µg
Coupling Phosphatase 1: 0.1 µg
Donor Substrate: 10 nmol
Acceptor Substrate: 10 nmol
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human N-Acetylglucosaminyltransferase 3/MGAT3 CF
N-glycosyl-oligosaccharide-glycoprotein N-acetylglucosaminyltransferase III
Mannosylglycoprotein N-acetyl-glucosaminyltransferase 3 (MGAT3), also known as GnT-III, regulates the branching of N-glycans. By transferring a GlcNAc residue to the beta -linked mannose of the trimannosyl core of N-linked oligosaccharides MGAT3 produces a bisecting GlcNAc structure (1). Bisecting GlcNAc is involved in a number of biological events, including the suppression of metastasis of cancer cells through modification on integrins (2, 3, 4) and the reduction of hepatitis B virus replication in hepatoma cells (5). The effect of the bisecting GlcNAc on a cancer cell counteracts that of the beta 1,6-GlcNAc created by MGAT5 (4, 6). Although MGAT3 and MGAT5 are functionally related, there is no significant homology at their primary sequences. In addition, bisecting GlcNAc on human Ig antibodies enhances their effector functions (7). Transgenic mice over expressing MGAT3 had aberrant glycosylation on apolipoprotein B and developed fatty livers (8). The enzymatic activity of recombinant human MGAT3 was determined using a phosphatase-coupled glycosyltransferase assay (9).
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