详细说明
- Purity>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
- Endotoxin Level<0.1 EU per 1 μg of the protein by the LAL method.
- ActivityMeasured by its ability to transfer Neu5Ac from CMP-Neu5Ac to alpha -lactose. The specific activity is >5 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
- SourceChinese Hamster Ovary cell line, CHO-derived Thr87-His414, with an N-terminal 6-His tag
- Accession #
- N-terminal Sequence
AnalysisHis - Predicted Molecular Mass38 kDa
- SDS-PAGE45-60 kDa, reducing conditions
| 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.
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Assay Procedure
Materials
- 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 # )
- 96-well Clear Plate (Catalog # )
- 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).
- References:
- 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.
- Long Name:ST3 beta-Galactoside alpha-2,3-Sialyltransferase 5
- Entrez Gene IDs:8869 (Human); 20454 (Mouse); 83505 (Rat)
- Alternate Names:3S-T; alpha 2,3-sialyltransferase V; CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase; EC 2.4.99.9; Ganglioside GM3 Synthase; GM3 synthase; lactosylceramide alpha-2,3-sialyltransferase; SATI; sialyltransferase 9 (CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase;GM3 synthase); Sialyltransferase 9; Siat9; SIATGM3S; ST3 beta-galactoside alpha-2,3-sialyltransferase 5; ST3Gal V; ST3GAL5; ST3GALV; ST3GalVSIAT9SATI
400-6226-992