Recombinant Human Lysosomal alpha-Glucosidase/GAA, CF

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

• Assay Buffer: 0.1 M Sodium Acetate, pH 4.5
• Recombinant Human Lysosomal alpha -Glucosidase (rhGAA) (Catalog # 8329-GH)
• Stop Solution: 4.4 mM Dinitrosalicylic Acid, 1 M Potassium Tartrate, 0.4 M Sodium Hydroxide in deionized water
• Standard: Maltose (Sigma, Catalog # M5885), 20 mM stock in deionized water
• Substrate: Starch from potato (Sigma, Catalog # 85642), 2% (w/v) stock in deionized water
• 96-well Clear Plate (Catalog # DY990)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 20 mM Maltose standard by adding 200 µL of the 20 mM Maltose Standard to 600 µL of Assay Buffer for a 5 mM stock. This is the first point of the standard curve.
2. Prepare the standard curve by performing five one-half serial dilutions of the 5 mM Maltose stock in Assay Buffer. Make sure there are 400 μL in each tube for each point of the curve (remove 400 μL from the last point of the curve). Prepare one tube with only 400 μL of Assay Buffer for the curve blank. The standard curve has a range of 19.5 to 625 nmol per well.
3. Dilute rhGAA to 32 ng/μL in Assay Buffer.
4. Dilute 2% starch to 1% in Assay Buffer by combining equal volumes of 2% starch and Assay Buffer.
5. Prepare reactions by combining 20 μL of diluted rhGAA with 380 μL of 1% starch (step 4). Include a control by combining 20 μL of Assay Buffer with 380 μL of 1% starch.
6. Vortex, spin, and then incubate reactions, control and standard curve at 37 °C for 1 hour.
7. Add 400 uL of Stop Solution to all vials, including standard curve.
8. Heat all vials at 95-100 °C for 6 minutes. Then, cool on ice. Tip: Use lid-locks on vials when heating.
9. Load 250 µL of each dilution of the standard curve, reactions and controls to empty wells in triplicate in plate.
10. Read plate at 546 nm (absorbance) in endpoint mode.
11. Calculate specific activity:

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

• rhGAA: 0.200 µg
• Starch: 0.475%