Recombinant Human Galactosylceramidase/GALC Protein, CF

• Assay Buffer: 50 mM Sodium Citrate, 125 mM NaCl, 0.5% Triton® X-100, pH 4.5
• Stop Solution: 0.5 M Glycine, 0.3 M NaOH (~pH 10.0)
• Recombinant Human Galactosylceramidase/GALC (rhGALC) (Catalog # 7310-GH)
• Substrate: 4-methylumbelliferyl-beta -D-galactopyranoside (Sigma, Catalog # M1633), 10 mM stock in DMSO
• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhGALC to 0.06 ng/μL in Assay Buffer.
2. Dilute Substrate to 1 mM in Assay Buffer.
3. Load in a plate 25 μL of 0.06 ng/μL rhGALC, and start the reaction by adding 25 μL of 1 mM Substrate.  Include a Substrate Blank containing 25 μL of Assay Buffer and 25 μL of 1 mM Substrate.
4. Seal plate and incubate at 37 °C for 20 minutes.
5. After incubation, stop the reactions by adding 50 μL of Stop Solution to each well.
6. Read at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in endpoint mode.
7. Calculate specific activity: 

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-methylumbelliferone (Sigma, Catalog # M1381).

• rhGALC: 0.0015 μg
• Substrate: 0.25 mM