Recombinant Human B4GAT1 His-tag Protein, CF
Recombinant Human B4GAT1 His-tag Protein, CF Summary
Product Specifications
His37-Cys415, with N-terminal 6-His tag
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.
6664-GT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| 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
- Glycosyltransferase Activity Kit (Catalog # EA001)
- Assay Buffer: 25 mM Tris, 10 mM CaCl2, 10 mM MnCl2 (supplied in kit), pH 7.5
- Recombinant Human beta -1,4-Glucuronyltransferase 1/B4GAT1 His-tag (rhB4GAT1) (Catalog # 6664-GT)
- Donor Substrate: UDP-GlcA (Sigma, Catalog # U5625), 10 mM stock in deionized water
- Acceptor Substrate: Xylose (V-lab, Catalog # BX53), 100 mM stock in deionized water
- 96-well Clear Plate (Catalog # DY990)
- 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.
- Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare reaction mixture containing 0.8 mM UDP-GlcA, 40 mM Xylose, and 4 µg/mL Coupling Phosphatase I in Assay Buffer.
- Dilute rhB4GAT1 to 20 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 20 µg/mL rhB4GAT1 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to all wells, excluding the standard curve and curve blank.
- 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 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:- rhB4GAT1: 0.5 µg
- Coupling Phosphatase I: 0.1 µg
- Xylose: 20 mM
- UDP-GlcA: 0.4 mM
Reconstitution Calculator
Background: beta-1,4-Glucuronyltransferase 1/B4GAT1
B4GAT1 was previously described in the literature as B3GNT1 (1). It is now characterized as a beta 1,4 glucuronyltransferase that is responsible for the synthesis of a glucuronyl-beta 1,4-xylosyl disaccharide found on alpha -dystroglycan ( alpha - DG), a peripheral membrane protein that binds to several extracellular matrix components (2). Proper glycosylation of alpha -DG is critical to maintain structural integrity and force transmission between the cytoskeleton and the extracellular matrix for efficient signal transduction. Mutation of B4GAT1 will lead to failure of proper glycosylation of alpha -DG and loss of receptor binding of alpha -DG, therefore causes congenital muscular dystrophies (CMDs) (3). The enzymatic activity of recombinant human B4GAT1 was determined using a phosphatase-coupled assay (4) using xylose as acceptor substrate.
- Sasaki, K. et al. (1997) PNAS 94:14294.
- Willer, T. et al. (2014) eLife; 3: e03941.
- Barresi, R. and Campbell, K.P. (2006) J. Cell Sci. 119:199.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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