Recombinant Human ST6GALNAC5 Protein, CF
Recombinant Human ST6GALNAC5 Protein, CF Summary
Product Specifications
Gly30-Phe336, with an N-terminal 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.
6715-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
- Sialyltransferase Activity Kit (Catalog # EA002)
- Assay Buffer: 25 mM MES, 20 mM MgCl2, pH 6.5
- Recombinant Human GalNAc alpha -2,6-sialyltransferase V/ST6GALNA (rhST6GALNAC5) (Catalog # 6715-GT)
- CMP-Neu5Ac (Sigma, Catalog # C8271), 10 mM stock in deionized water
- Fetuin (Sigma, Catalog # F3385), 50 mg/mL 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 Sialyltransferase Activity 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.
- Complete the standard curve by performing six one-half serial dilutions of the 100 μM Phosphate stock using Assay Buffer. The standard curve has a range of 0.078 to 5 nmoles per well.
- Dilute CMP-Neu5Ac to 1.5 mM in Assay Buffer.
- Dilute Fetuin to 30 mg/mL in Assay Buffer.
- Dilute Coupling Phosphatase 2 to 12 μg/mL in Assay Buffer.
- Prepare reaction mixture by combining equal volumes of diluted CMP-Neu5Ac, Fetuin, and Coupling Phosphatase 2.
- Dilute rhST6GALNAC5 to 10 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 10 µg/mL rhST6GALNAC5 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 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:- rhST6GALNAC5: 0.25 μg
- Coupling Phosphatase 2: 100 ng
- CMP-Neu5Ac: 250 µM
- Fetuin: 250 μg
Reconstitution Calculator
Background: ST6 GalNAc alpha-2,6-sialyltransferaseV/ST6GALNAC5
Gangliosides are acidic glycosphingolipids that contain one or more sialic acid residues (1). They are abundant in the nervous system, where they play crucial modulatory roles in cellular recognition, interaction, adhesion, and signal transduction, particularly during early developmental stages. The expression of gangliosides in the nervous system is developmentally regulated through various sialyltransferases (2). ST6GALNAC5 is a sialyltransferase involved in the biosynthesis of ganglioside GD1a (NeuAc alpha 2,3Gal beta 1,3GalNAc beta 1,4(NeuAc alpha 2,3)Gal beta 1,4Glc beta 1-Cer) from GM1b (NeuAc alpha 2,3Gal beta 1,3GalNAc beta 1,4Gal beta 1,4Glc beta 1-Cer), and its expression is restricted to the brain (3, 4). ST6GALNAC5 has been identified as a key player in the metastasis of breast cancer cells to the brain by potentially enabling the cancer cells to cross the blood-brain barrier (5, 6). The recombinant ST6GALNAC5 was active on fetuin from fetal calf serum when assayed using a phosphatase-coupled method (7) suggesting the substrate specificity of ST6GALNAC5 may require further characterization.
- Kolter, T. et al. (2002) J. Biol. Chem. 277:25859.
- Yu, R.K. et al. (2008) Glycoscience DOI: 10.1007/978-3-540-30429-6_41.
- Okajima, T. et al. (1999) J. Biol. Chem.274:30557.
- Harduin-Lepers, A. et al. (2005) Glycobiology 15:805.
- Bos, P.D. et al. (2009) Nature 459:1005.
- Arshad, F. et al. (2011). Patholog. Res. Int. Doi: 10.4061/2011/920509.
- Wu, Z.L. et al. (2010) Glycobiology doi: 10.1093/glycob/cwq187.
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