Recombinant Human ASAH2 Protein, CF

Catalog #: 3557-AH Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
3557-AH-010

R&D Systems Recombinant Proteins and Enzymes

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All ASAH2/N-acylsphingosine Amidohydrolase-2 Proteins and Enzymes

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Human ASAH2 Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to hydrolyze the substrate C12:0 ceramide into sphingosine and dodecanoic acid. The specific activity is >5,000 pmol/min/µg, as measured under the described conditions.

Source

Chinese Hamster Ovary cell line, CHO-derived human ASAH2/N-acylsphingosine Amidohydrolase-2 protein
Thr15-Ile761, with an N-terminal 6-His tag

Accession #

AAF86240

N-terminal Sequence
Analysis

His

Predicted Molecular Mass

83 kDa

SDS-PAGE

95-140 kDa, reducing conditions

Product Datasheets

3557-AH

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

3557-AH

Formulation	Supplied as a 0.2 μm filtered solution in MES, NaCl and Sodium Cholate.
Shipping	The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

Assay Buffer: 25 mM MES, 150 mM NaCl, 1% Sodium Cholate, pH 6.5
o-PA Buffer: 0.2 M NaOH, 0.1% beta -mercaptoethanol (v/v)
Recombinant Human ASAH2/N‑acylsphingosine Amidohydrolase-2 (rhASAH2) (Catalog # 3557-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

Dissolve 10 µL of C12-ceramide stock in 1.99 mL Assay Buffer for a 250 µM Substrate concentration. (Note: Preheat assay buffer to 37 °C and vortex for 30 seconds to dissolve Substrate).
Dilute rhASAH2 to 0.05 µg/mL in Assay Buffer.
Combine 200 µL of 250 µM Substrate and 50 µL of 0.05 µg/mL rhASAH2. Include one blank containing 50 µL rhASAH2 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.
Load 200 µL 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).

Per Well:

rhASAH2: 0.001 µg
C12-ceramide: 100 μM
o-PA: 1 mg/mL

Reconstitution Calculator

Background: ASAH2/N-acylsphingosine Amidohydrolase-2

The human ASAH2 gene encodes N-acylsphingosine amidohydrolase-2, also known as neutral or non-lysosomal ceramidase. ASAH2 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). An N-terminally truncated form of human ASAH2 has been shown to localize to mitochondria (4). A major physiological function of ASAH2 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).

References