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Recombinant Mouse CHST1 Protein, CF 20 UG图1

Recombinant Mouse CHST1 Protein, CF 20 UG

2024-11-24 20:08IP属地 广东省东莞市 电信00留言

5355-ST

 

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.      

  • 6 months from date of receipt, -70 °C as supplied.

  • 3 months, -70 °C under sterile conditions after opening.


Assay Procedure

Materials

  1. 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.

  2. 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.

  3. Prepare reaction mixture containing 0.286 mM PAPS, 0.214 M alpha -lactose, and 28.57 μg/mL Coupling Phosphatase 3 in Assay Buffer.

  4. Dilute rmCHST1 to 15 µg/mL in Assay Buffer.

  5. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.

  6. Load 15 µL of the 15 µg/mL rhCHST1 into the plate. Include a Control containing 15 µL of Assay Buffer.

  7. Add 35 µL of reaction mixture to the wells, excluding the standard curve.

  8. Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.

  9. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.

  10. Add 100 µL of deionized water to all wells. Mix briefly.

  11. Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.

  12. Read plate at 620 nm (absorbance) in endpoint mode.

  13. 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 constant is 0.49 in the presence of the final assay condition of 0.5 M NaCl.

Per Reaction:

Background: Carbohydrate Sulfotransferase 1/CHST1

The mouse CHST family is comprised of 13 enzymes. 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). Mouse CHST1 has 94% amino acid sequence identity with the human ortholog. The activity of the recombinant mouse CHST1 is measured using a PAP-specific phosphatase-coupled sulfotransferase assay (5).

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