Recombinant Mouse ST3GAL5/GM3 Synthase Protein, CF
Recombinant Mouse ST3GAL5/GM3 Synthase Protein, CF Summary
Product Specifications
Thr87-His414, with an N-terminal 6-His tag
Analysis
Product Datasheets
8454-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.
8454-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
- Assay Buffer: 25 mM MES, pH 7.0
- Recombinant Mouse ST3GAL5/GM3 Synthase (rmST3GAL5) (Catalog # 8454-GT)
- Donor Substrate: CMP-Sialic Acid (CMP-Neu5Ac) (Sigma, Catalog # C8271), 10 mM stock in deionized water
- Acceptor Substrate: alpha -Lactose (Sigma, Catalog # L2643), 300 mM stock in deionized water
- Sialyltransferase Activity Kit (Catalog # EA002)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute 1 mM Phosphate Standard provided by the Sialyltransferase Activity 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 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 0.4 mM CMP-Sialic Acid, 80 mM alpha -lactose, and 2 µg/mL Coupling Phosphatase 2.
- Dilute rmST3GAL5 to 40 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 40 µg/mL rhST6GAL1 into the plate. Include a Control containing 25 µL of Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Cover the plate with parafilm or a plate sealer and incubate at room temperature for 3 hours.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 100 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 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.
- rmST3GAL5: 1.0 µg
- Coupling Phosphatase 2: 0.05 µg
- CMP-Sialic Acid: 0.2 mM
- alpha -Lactose: 40 mM
Background: ST3GAL5/GM3 Synthase
Sialyltransferases add sialic acid to glycoproteins or glycosphingolipids and play important roles in many biological processes including immune recognition, pathogen infection, and cell adhesion (1). ST3GAL5 is a type II membrane protein localized in the trans-Golgi network that can transfer sialic acid to lactosylceramide (LacCer) to form GM3 (NeuAc alpha 2-3Gal beta 1-Glc beta 1-Ceramide), the main ganglioside in human plasma (2). GM3 is known to participate in differentiation, proliferation and maintenance of fibroblast morphology (3). It also plays role in signal transduction, and integrin-mediated cell adhesion. Mutation of ST3GAL5 has been associated with Amish infantile epilepsy syndrome (4) and type 2 diabetes (5). Despite the ubiquitous distribution of ganglioside GM3 in human tissues, the expression of ST3GAL5 is found in a tissue-specific manner, with predominant expression in brain, skeletal muscle, and testis, and very low expression in liver (2). The enzymatic activity of recombinant mouse ST3GAL5 was determined using a phosphatase-coupled glycosyltransferase assay (6).
- Varki, A. (1999) Glycobiology 2:25.
- Ishii, A. et al (1998) J. Biol. Chem. 273:31652.
- Uemura, S. et al (2009) Mol. Biol. Cell 20:3088.
- Simpson, M. A. et al. (2004) Nat. Genet. 36:1225.
- Tagami, S. et al. (2002) J. Biol. Chem. 277:3085.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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