Arylsulfatase A/ARSA Protein, Mouse (HEK293, His)

Materials
Assay buffer: 50 mM NaOAc, 0.5 M NaCl, pH 4.5
Arylsulfatase A/ARSA Protein, Mouse (HEK293, His) (HY-P75499)
Substrate: 4-Nitrocatechol Sulfate (PNCS) , diluted to 100 mM in ddH₂O
Standard: 4-Nitrocatechol (PNC, HY-W066890)
Sodium Hydroxide (NaOH) : 2.0 M stored in deionized water
96-well transparent plate
Microplate reader

Procedure
1. Standard Curve: Prepare PNC at concentrations of 0, 0.078125×10⁶, 0.15625×10⁶, 0.3125×10⁶, 0.625×10⁶, 1.25×10⁶, 2.5×10⁶, 5×10⁶ pmol. Measure the OD₅₁₀ for each concentration. Plot the standard curve with standard concentration (pmol) on the x-axis and the OD₅₁₀ on the y-axis.
2. Dilute Mouse ARSA in assay buffer to concentrations of 2, 20, 200 μg/mL.
3. Dilute the substrate to 2 mM in assay buffer.
4. Experimental group: Mix 75 μL of 2, 20, 200 μg/mL Mouse ARSA with 75 μL of 2 mM substrate.
Blank Control: Combine 75 μL of assay buffer with 75 μL of substrate.
Set up three replicates for each group and add to 1.5 mL centrifμge tubes.
5. Incubate in a water bath at 37°C for 1 hour.
6. Add 150 μL of 0.2 M NaOH to stop the reaction.
7. Transfer 200 μL to a transparent 96-well plate.
8. Read the absorbance at 510 nm in endpoint mode.
9. Calculate specific activity: