Measured by its ability to transfer sulfate from PAPS to alpha -Lactose. The specific activity is >500 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human Carbohydrate Sulfotransferase 1/CHST1 protein Arg24-Ser411, with an N-terminal 6-His tag
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Enzyme Activity
Theoretical MW
45 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
58 kDa, reducing conditions
Publications
Read Publications using 5316-ST in the following applications:
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute 1 mM Phosphate Standard provided by the Universal Sulfotransferase 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 PAPS, 200 mM alpha -lactose, and 40 μg/mL Coupling Phosphatase 3 in Assay Buffer.
Dilute rhCHST1 to 6 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 6 µg/mL rhCHST1 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 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) x coupling rate**
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control. **Coupling rate is 0.49 for conditions described.
Per Reaction:
rhCHST1: 0.15 μg
Coupling Phosphatase 3: 1.0 μg
PAPS: 0.2 mM
alpha –lactose: 100 mM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human CHST1 Protein, CF
The CHST family is comprised of 14 enzymes in humans. All members of this family are Golgi-localized type II membrane proteins. Only the luminal and enzymatic domain is expressed in each of our recombinant CHST proteins. These enzymes transfer sulfate (i.e., sulfonate) onto the 6-O or 4-O positions of GalNAc, Gal and GlcNAc residues on glycoproteins, proteoglycans and glycolipids (1). This sulfation often creates specific epitopes that can be recognized by extracellular matrix proteins, cell surface receptors and viruses (2). CHST1, also known as keratan sulfate Gal-6 sulfotransferase, transfers sulfate to position 6 of galactose residues on keratan sulfate (3). It also has sulfotransferase activity on sialyl N-acetyllactosamine structures and participates in biosynthesis of selectin ligands that play a central role in lymphocyte homing at sites of inflammation (4). Human CHST1 shares 94% amino acid sequence identity with mouse CHST1.
The activity of the
recombinant human CHST1 is measured using a PAP-specific phosphatase-coupled
sulfotransferase assay (5).
Hemmerich, S. and S.D. Rosen (2000) Glycobiology 10:849.
Bowman, K.G. and C.R. Bertozzi (1999) Chem. Biol. 5:447.
Fukuta, M. et al. (1997) J. Biol. Chem. 272:32321.
Reviews for Carbohydrate Sulfotransferase 1/CHST1/KS6ST (5316-ST) (0)
There are no reviews for Carbohydrate Sulfotransferase 1/CHST1/KS6ST (5316-ST).
By submitting a review you will receive an Amazon e-Gift Card or Novus Product Discount.
Review with no image -- $10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen
Review with an image -- $25/€18/£15/$25 CAD/¥150 Yuan/¥2500 Yen
FAQs for Carbohydrate Sulfotransferase 1/CHST1/KS6ST (5316-ST) (0)
The concentration calculator allows you to quickly calculate the volume, mass or concentration of your vial. Simply enter your mass, volume, or concentration values for your reagent and the calculator will determine the rest.
=
÷
Review this Product
Be the first to review our Recombinant Human CHST1 Protein, CF and receive a gift card or discount.