Recombinant Mouse CHST4 Protein, CF

• 6 months from date of receipt, -20 to -70 °C as supplied.
• 3 months, -20 to -70 °C under sterile conditions after opening.

• Assay Buffer: 0.1 M Tris, 30 mM MgCl₂, pH 7.5
• Recombinant Mouse Carbohydrate Sulfotransferase 4/CHST4 (rmCHST4) (Catalog # 5547-ST)
• Donor Substrate: 3′-Phosphoadenosine-5′-phosphosulfate/PAPS (PAPS) (Catalog # ES019)
• Acceptor Substrate: N-acetyl-alpha -D-glucosamine (GlcNAc)  (Calbiochem, Catalog #1079), 1 M stock in deionized water
• Universal Sulfotransferase Activity Kit (Catalog # EA003)
• 96-well Clear Plate
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard 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.
2. 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.
3. Prepare reaction mixture by containing 0.286 mM PAPS, 0.643 M GlcNAc, 0.0143 mg/mL Coupling Phosphatase 3.
4. Dilute rmCHST4 to 40 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 40 µg/mL rmCHST4 into the plate. Include a Substrate Blank 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:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

• rmCHST4:  0.6 µg
• Coupling Phosphatase 3: 0.5 μg
• PAPS: 0.2 mM
• GlcNAc: 450 mM