Recombinant Human ST6GALNAC4 Protein, CF
Recombinant Human ST6GALNAC4 Protein, CF Summary
Thr36-Arg302, with a C-terminal 6-His tag
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.
|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 Buffer: 50 mM Tris, 10 mM MnCl2 (supplied in kit), pH 7.0
- Recombinant Human ST6GALNAC4 (rhST6GALNAC4) (Catalog # 6876-GT)
- Donor Substrate: CMP-Sialic Acid (Sigma, Catalog # C8271), 10 mM stock in deionized water
- Acceptor Substrate: Fetuin (Sigma, Catalog # F3385), 50 mg/mL stock in diH20
- Sialyltransferase Activity Kit (Catalog # EA002)
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute CMP-Sialic Acid to 2.5 mM in Assay Buffer.
- Dilute Coupling Phosphatase 2 to 10 µg/mL in Assay Buffer.
- Prepare reaction mixture by combining 75 µL of 2.5 mM CMP-Sialic Acid, 150 µL of 10 µg/mL Coupling Phosphatase 2, 75 µL of 50 mg/mL Fetuin, and 75 µL of Assay Buffer.
- Dilute rhST6GALNAC4 to 0.8 µg/mL in Assay Buffer.
- Dilute 1 mM Phosphate Standard by adding 40 µL to 360 µL of Assay Buffer for a 100 μM stock. This is the first point of the standard curve.
- Continue 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 5.0 nmol per well.
- 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 0.8 µg/mL rhST6GALNAC4 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 ºC for 20 minutes.
- Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- 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 the control.Per Reaction:
- rhST6GALNAC4: 0.02 µg
- Coupling Phosphatase 2: 0.1 µg
- Fetuin: 250 µg
- CMP-Sialic Acid: 0.25 mM
ST6GALNAC4 is a type II membrane protein that catalyzes the transfer of sialic acid from CMP-sialic acid to galactose-containing substrates (1). This enzyme works on both glycoproteins and glycolipids and has strict substrate specificity, utilizing only the trisaccharide sequence Neu5Ac alpha 2-3Gal beta 1-3GalNAc of glycoproteins and glycolipids (2). In particular, this enzyme is involved in the synthesis of ganglioside GD1A from GM1B (3). GD1A is the target pathogenic antigen in the autoimmune disease, Guillain-Barre Syndrome (4, 5). The activity of this enzyme has been measured using a phosphatase-coupled method (6).
- Harduin-Lepers, A. et al. (2005) Glycobiology 15:805.
- Harduin-Lepers, A. et al. (2000) Biochem. J. 352:37.
- Lee, Y. C. et al. (1999) J. Biol. Chem. 274:11958.
- Kusunoki, S. et al. (1994) Ann. Neurol. 35:570.
- Goodfellow, J.A. et al. (2005) J. Neurosci. 25:1620.
- Wu, Z.L. et al. (2011) Glycobiology 21:727.
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Glycosyltransferase Activity Assays
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