Alpha‑Galactosidase Activity Assay Kit (Fluorometric)

Alpha-Galactosidase (α-Gal) hydrolyzes alpha-galactosyl moieties found in glycolipids and glycoproteins. In mammals, α-Gal hydrolyzes poly- and oligosaccharides commonly found in dietary sources that are difficult to digest. Therefore, α-Gal is used in dietary supplements that help to reduce the production of intestinal gases due to consumption of certain foods. It is known total α-Gal activity is due to two major isozymes with unique, yet different thermostability profiles. Alpha-Galactosidase A, is thermolabile and represents approximately 90% of total α-Gal activity. Fabry Disease, a lysosomal disease disorder, is characterized by mutations in alpha-Galactosidase A. These mutations cause abnormal accumulation of glycosphingolipids in lysosomes.

Alpha‑Galactosidase Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring the activity of alpha‑Galactosidase in cell lysates, plasma, serum, urine, saliva and other biological liquid samples. In this assay, alpha‑Galactosidase cleaves a synthetic specific substrate releasing a fluorophore. The fluorescence signal is measured at Ex/Em = 360/445 nm. The increase in fluorescence intensity is directly proportional to alpha‑Galactosidase activity in the sample.

• Microplate reader capable of measuring fluorescence intensity at Ex/Em = 360/445 nm
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Incubator at 37°C
• Tubes to prepare sample dilutions
• 15 mL and 50 mL conical tubes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 40 µL of samples, positive control, substrate background to the 96-well microtiter plate (black bottom).
2. Add 60 µL of standard to the 96-well microtiter plate.
3. Add 20 µL diluted α-Gal substrate solution to each well, but not the standard wells. Carefully shake the plate for a few seconds to mix.
4. Incubate at 37°C for 30 minutes with mixing, protect from light.
5. Add 200 µL Stop Solution. Put the plate on a shaker for 2 minutes to mix well (protect from light).
6. Read the fluorescence signal with a fluorescence plate reader at Ex/Em = 360/445 nm.