Recombinant Human ST6GALNAC1 Protein
Recombinant Human ST6GALNAC1 Protein Summary
Learn more about Fluorescent Glycan Labeling and DetectionProduct Specifications
Lys36-Asn600, with a C-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.
9154-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)
- 10X Assay Buffer (supplied in kit): 250 mM Tris, pH 7.5
- Recombinant Human ST6 Sialyltransferase 1/ST6GALNAC1 (rhST6GALNAC1) (Catalog # 9154-GT)
- CMP-Sialic acid (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
- Prepare 1X Assay Buffer containing 10 mM MnCl2 by combining 10X stocks and diluting 10 fold with deionized water.
- 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 1X Assay Buffer for a 100 µM stock.
- Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in 1X Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.
- Prepare reaction mixture containing 0.5 mM CMP-Sialic Acid, 20 mg/mL Fetuin and 4 µg/mL Coupling Phosphatase 2 in 1X Assay Buffer.
- Dilute rhST6GALNAC1 to 20 µg/mL in 1X Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of 1X Assay Buffer.
- Load 25 µL of the 20 µg/mL rhST6GALNAC1 into the plate. Include a control containing 25 µL of 1X Assay Buffer.
- Add 25 µL of reaction mixture to the wells, excluding the standard curve.
- Cover the plate with 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:- rhST6GALNAC1: 0.5 µg
- CMP-Sialic Acid: 250 µM
- Fetuin: 500 µg
- Coupling Phosphatase 2: 0.1 µg
Reconstitution Calculator
Background: ST6 Sialyltransferase 1/ST6GALNAC1
Sialic acid molecules attached to glycoproteins or glycosphingolipids play important roles in various biological processes such as immune recognition, pathogen infection, and cell adhesion. Sialyltransferases are key enzymes that regulate the serum levels of sialic acid‑containing molecules. Alpha‑N‑acetylgalactosaminide alpha ‑2,6‑sialyltransferase 1, or ST6GALNAC1, is a type II membrane protein localized in the Golgi network and catalyzes 2,6‑sialylation of the Tn antigen (GalNAc‑O‑Ser/Thr) (1, 2). Sialyl‑Tn antigen is highly expressed in several human carcinomas and is associated with carcinoma aggressiveness and poor prognosis (3). Breast cancer cells transfected with ST6GALNAC1 synthesized the sialyl‑Tn antigen structure (4). The enzyme may also be used to synthesize a cancer vaccine (5). The activity of this enzyme has been measured using a phosphatase‑coupled assay (6).
- Ikehara, Y, et al. (1999) Glycobiology 9:1213.
- Sewell, R. et al. (2006) J. Biol. Chem. 281:3586.
- Lima, L. et al. (2013) Br. J. Cancer 109:2106.
- Julien, S. et al. (2001) Glycoconj. J. 18:883.
- Julien, S. et al. (2009) Br. J. Cancer. 100:1746.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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