Recombinant Human ASAHL Protein, CF Summary
Met1-Lys359 (Val151Ile), with a C-terminal 10-His tag
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.
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.
|Formulation||Supplied as a 0.2 μm filtered solution in MES, NaCl and Glycerol.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 0.1 M NaOAc, 0.1% (v/v) NP-40 substitute (Fluka, Catalog # 74385), pH 4.0
- Recombinant Human ASAHL/N‑acylethanolamine-hydrolyzing Acid A (rhASAHL) (Catalog # 4494-AH)
- Palmitoyl Ethanolamide (PEA) (Tocris, (Tocris, Catalog # 0879), 25 mM stock in dimethyl formamide
- Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
- NaOH (Sigma, Catalog # 221465), 2 M stock in deionized water
- beta -mercaptoethanol (Sigma, Catalog # M-7154)
- 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
- Dilute PEA to 50 µM in Assay Buffer by dissolving 10 µL of 25 mM stock in 4.99 mL of Assay Buffer (Note: Preheat assay buffer to 37 °C and vortex for 30 seconds to completely solubilize the PEA).
- Dilute rhASAHL to 1.25 µg/mL in Assay Buffer.
- Combine 200 µL of 50 µM PEA, 50 µL of 1.25 µg/mL rhASAHL, and 2.5 µL of 1 M DTT.
- Incubate reaction tubes at 37 °C for 1 hour.
- Dilute NaOH to 0.2 M in deionized water.
- Combine 3.84 mL of 0.2 M NaOH with 4 µL beta -mercaptoethanol and 160 µL of 50 mg/mL o-PA.
- Stop the reactions by adding 250 µL of the o-PA mixture (step 6) to all the vials and mix well.
- Incubate at room temperature for 10 minutes.
- Combine 250 µL of o-PA mixture, 50 µL of 1.25 µg/mL rhASAHL, and 200 µL of 50 µM PEA in this order for a control.
- Load 200 µL of reaction mixtures and control in a plate.
- Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
- Calculate specific activity (Average duplicates):
Specific Activity (pmol/min/µg) =
|Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU)|
|Incubation time (min) x amount of enzyme (µg)|
*Adjusted for Control
**Derived using calibration standard ethanolamine (Sigma, Catalog # E9508).
- rhASAHL: 0.025 µg
- Palmitoyl Ethanolamide: 20 µM
- o-PA: 1 mg/mL
Background: ASAHL/N-acylethanolamine-hydrolyzing Acid Amidase
The human NAAA gene encodes N-acylethanolamine-hydrolyzing acid amidase, also known as ASAH-like protein (ASAHL), a fatty acid amidase with maximal activity at acidic pH (1). NAAA hydrolyzes a number of N-acyl ethanolamines, including N-myristoyl-, N-stearoyl, N-oleoyl, and N-arachidonoyl, but is most active against N‑palmitoylethanolamine (2). NAAA is a member of the cholylglycine hydrolase family of enzymes, and is structurally similar to acid ceramidase (3). NAAA is both a lysosomal and a secreted enzyme, and like acid ceramidase, has been observed to be proteolytically processed during maturation (3). NAAA can be distinguished from anandamide amidohydrolase by its lack of inhibition by methyl arachidonyl fluorophosphonate (2).
- Hong, S.B. et al. (1999) Genomics 62:232.
- Ueda, N. et al. (2001) J. Biol. Chem. 276:35552.
- Tsuboi, K. et al. (2005) J. Biol. Chem. 280:11082.
No product specific FAQs exist for this product, however you mayView all Proteins and Enzyme FAQs
Reviews for Recombinant Human ASAHL Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Human ASAHL Protein, CF and earn rewards!
Have you used Recombinant Human ASAHL Protein, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥1250 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image