>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
<1.0 EU per 1 μg of the protein by the LAL method.
81 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dissolve 10 µL of 50 mM stock of Substrate in 1.99 mL Assay Buffer for a 250 µM concentration. (Note: Preheat assay buffer to 37 °C and vortex for 30 seconds to dissolve Substrate).
Dilute rmASAH2 to 0.05 µg/mL in Assay Buffer.
Combine 200 µL of 250 µM Substrate and 50 µL of 0.05 µg/mL rmASAH2. Include one blank containing 50 µL rmASAH2 and 200 µL Assay Buffer and another blank containing 200 µL Substrate and 50 µL Assay Buffer.
Incubate at 37 °C for 1 hour.
Stop reactions by heating them at 95-100 °C for 5 minutes.
Dilute o-PA to 2 mg/mL in o-PA Buffer.
Add 250 µL of the o-PA mixture to all reaction vials, including controls. Mix well.
Incubate at room temperature for 10 minutes. Note: It is important not to deviate from this incubation time.
Load 200 µL (in duplicate) of reaction mixtures and controls in a plate.
Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
Calculate specific activity using the following equation:
Specific Activity (pmol/min/µg) =
Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)
Incubation time (min) x amount of enzyme (µg)
*Average duplicates, use the control with the higher RFU value to adjust fluorescence. **Derived using calibration standard Sphingosine (Avanti Polar Lipids, Catalog # 860490P).
rmASAH2: 0.001 µg
C12-ceramide: 100 μM
o-PA: 1 mg/mL
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Mouse ASAH2 Protein, CF
The mouse ASAH2 gene encodes acylsphingosine amidohydrolase-2, also known as neutral ceramidase. Neutral ceramidase is a type II integral membrane protein that can be cleaved to produce a soluble secreted protein (1). The enzyme is abundant in the brush border membranes of the intestine, but is also expressed in tissues such as kidney, brain and liver (2, 3). A major physiological function of neutral ceramidase is the metabolism of dietary sphingolipids, but the enzyme may also be involved in the generation of messenger molecules such as sphingosine and sphingosine 1-phosphate (3).
Tani, M. et al. (2003) J. Biol. Chem. 278:10523.
Kono, M. et al. (2006) J. Biol. Chem. 281:7324.
Mitsutake, S. et al. (2001) J. Biol. Chem. 276:26249.
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