Recombinant Human B3GAT3 Protein, CF
Recombinant Human B3GAT3 Protein, CF Summary
Product Specifications
Glu72-Val335, with an N-terminal Met and 6-His tag
Analysis
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.
6808-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with dry ice or equivalent. 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, 1 mM MnCl2 (supplied in kit), pH 7.0
- Buffer B: 100 mM Tris, 5 mM CaCl2, pH 7.5
- Recombinant Human beta -1,3-Glucuronyltransferase 3/B3GAT3 (rhB3GAT3) (Catalog # 6808-GT)
- Substrate: UDP-GlcA (Sigma, Catalog # U5625), 10 mM stock in DMSO
- Glycosyltransferase Activity Kit (Catalog # EA001)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute the 1 mM Phosphate standard stock by adding 40 µL to 360 µL of Buffer A for a 100 µM stock. This is the first point of the standard curve.
- Prepare standard curve by performing six additional 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 Substrate to 1.25 mM in Buffer A.
- Dilute rhB3GAT3 to 80 µg/mL in 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 80 µg/mL rhB3GAT3 into the plate. Include 25 µL of Buffer A for a blank control.
- Start the reaction by adding 25 µL of Substrate to the wells, excluding the standard curve.
- Cover the plate with a plate sealer and incubate at 37 °C for 4 hours.
- Dilute Coupling Phosphatase 1 to 2 μg/mL in Buffer B.
- Add 50 µL of 2 µg/mL Coupling Phosphatase 1 to the reaction wells and blanks, excluding the standard curve. Add 50 µL of Buffer B to the standard curve.
- Tap to 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 Substrate Blank.
Per Reaction:- rhB3GAT3: 2 µg
- Coupling Phosphatase 1: 100 ng
- Substrate: 0.625 mM
Reconstitution Calculator
Background: beta-1,3-Glucuronyltransferase 3/B3GAT3
B3GAT3 is an essential enzyme involved in the synthesis of the linkage region of heparan sulfate and chondroitin sulfate proteoglycans (1). It transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcA) to the trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. B3GAT3 knockout mice are embryonic lethal before the 8-cell stage due to failed cytokinesis, suggesting critical roles for chondroitin sulfate and heparan sulfate in embryonic cell division (2). Unlike the broad substrate specificity exhibited by B3GAT1, B3GAT3 shows strict specificity for Gal beta 1-3Gal beta 1 (3). The crystal structure shows the enzyme is an alpha / beta protein with two subdomains that constitute the donor and acceptor binding sites with the active site lying in the cleft between the two subdomains (4). Like most known glycosyltransferases, B3GAT3 is a type II Golgi-resident transmembrane protein with a short N‑terminal cytoplasmic domain and a single-pass transmembrane domain followed by an enzymatic domain in the lumen of Golgi apparatus. In the current assay, hydrolase activity against UDP-GlcA was measured using a phosphatase-coupled method (5).
- Kitagawa, H. et al. (1998) J. Biol. Chem. 273:6615.
- Isumikawa, T. et al. (1998) J. Biol. Chem. 285:12190.
- Fondeur-Gelinotte, M. et al. (2007) Glycobiology 17:857.
- Pedersen, L.C. et al. (2000) J. Biol. Chem. 275:34580.
- Wu, Z.L, et al. (2011) Glycobiology 21:727.
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