Recombinant Mouse ASAH2 Protein, CF

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Product Details

Summary
Reactivity MuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Mouse ASAH2 Protein, CF Summary

Details of Functionality
Measured by its ability to hydrolyze the substrate C12:0 ceramide into sphingosine and dodecanoic acid. The specific activity is > 3,000 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived mouse ASAH2/N-acylsphingosine Amidohydrolase-2 protein
Thr34-Thr756, with an N-terminal 6-His tag
Accession # NP_061300
Accession #
N-terminal Sequence
His
Protein/Peptide Type
Recombinant Enzymes
Gene
Asah2
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
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.
SDS-PAGE
113 kDa, reducing conditions
Publications
Read Publications using
3558-AH in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in MES, NaCl and Sodium Cholate.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Assay Procedure
  • Assay Buffer: 25 mM MES, 150 mM NaCl, 1% (w/v) Sodium Cholate, pH 6.5
  • o-PA Buffer: 0.2 M NaOH, 0.1% beta -mercaptoethanol (v/v)
  • Recombinant Mouse ASAH2/N‑acylsphingosine Amidohydrolase-2 (rmASAH2) (Catalog # 3558-AH)
  • Substrate: C12-ceramide (Avanti Polar Lipids, Catalog # 860512P), 50 mM stock in Chloroform
  • o-Phthaldialdehyde (o-PA) (Sigma, Catalog # P0657), 50 mg/mL stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. 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).
  2. Dilute rmASAH2 to 0.05 µg/mL in Assay Buffer.
  3. 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.
  4. Incubate at 37 °C for 1 hour.
  5. Stop reactions by heating them at 95-100 °C for 5 minutes.
  6. Dilute o-PA to 2 mg/mL in o-PA Buffer.
  7. Add 250 µL of the o-PA mixture to all reaction vials, including controls. Mix well.
  8. Incubate at room temperature for 10 minutes. Note: It is important not to deviate from this incubation time.
  9. Load 200 µL (in duplicate) of reaction mixtures and controls in a plate.
  10. Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
  11. 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).

Per Well:
  • rmASAH2: 0.001 µg
  • C12-ceramide: 100 μM
  • o-PA: 1 mg/mL

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse ASAH2 Protein, CF

  • ASAH2 N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2
  • ASAH2
  • BCDase
  • HNAC1
  • LCDase
  • Nacylsphingosine Amidohydrolase2
  • NCDase
  • N-CDase

Background

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).

  1. Tani, M. et al. (2003) J. Biol. Chem. 278:10523.
  2. Kono, M. et al. (2006) J. Biol. Chem. 281:7324.
  3. Mitsutake, S. et al. (2001) J. Biol. Chem. 276:26249.

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Publications for ASAH2/N-acylsphingosine Amidohydrolase-2 (3558-AH)(2)

We have publications tested in 1 confirmed species: Mouse.

We have publications tested in 2 applications: Bioassay, In Vivo.


Filter By Application
Bioassay
(1)
In Vivo
(1)
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Mouse
(2)
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Bioinformatics

Gene Symbol Asah2
Uniprot