Recombinant Human Chitotriosidase/CHIT1 Protein, CF

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

• Assay Buffer: 10 mM MES, pH 6.0
• Reading Buffer: 100 mM Tris, pH 9.0
• Recombinant Human Chitotriosidase/CHIT1 (rhCHIT1) (Catalog # 3559-GH)
• Substrate: 4-Methylumbelliferyl beta -D-N,N’,N”-triacetylchitotriose (4-MU-TACT) (Sigma, Catalog # M‑5639), 5 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 rhCHIT1 to 0.4 ng/μL in Assay Buffer.
2. Dilute Substrate to 200 μM in Assay Buffer.
3. Mix equal volumes of 0.4 ng/μL rhCHIT1 and 200 μM Substrate. Include a Substrate Blank with equal volumes of Assay Buffer and Substrate.
4. Incubate at room temperature for 5 minutes. Protect from light.
5. Load 90 μL of Reading Buffer per sample in plate.
6. Add 10 μL of each reaction mixture into wells containing Reading Buffer.
7. Immediately read at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in endpoint mode.
8. Calculate specific activity:

     *Adjusted for Substrate Blank

     **Derived using calibration standard 4-Methylumbelliferone (4-MU) (Sigma, Catalog # M1381).

• rhCHIT1: 0.002 μg
• Substrate: 10 μM