ASAH2 Protein, Mouse (HEK293, His)

ASAH2 protein has N-acylsphingosine amide hydrolase activity, which is essential for lipid digestion and sphingolipid metabolism. It is present in different cellular compartments, highlighting its versatility. ASAH2 Protein, Mouse (HEK293, His) is the recombinant mouse-derived ASAH2 protein, expressed by HEK293 , with N-His labeled tag.

The ASAH2 protein is recognized for its role in enabling N-acylsphingosine amidohydrolase activity, a crucial function in lipid digestion and sphingolipid metabolic processes. Its presence in diverse cellular compartments, including caveola, extracellular space, and mitochondrion, underscores its versatility. As an integral component of the plasma membrane, ASAH2 holds significance in cellular structure and function. The orthologous relationship with several human genes, including ASAH2B (N-acylsphingosine amidohydrolase 2B), further emphasizes its evolutionary conservation. The expression profile reveals a broad presence across tissues, with notable levels in the small intestine adult and large intestine adult, suggesting a pivotal role in maintaining lipid homeostasis and metabolic processes in various physiological contexts.

Measured by its ability to hydrolyze the substrate C12:0 ceramide into sphingosine and dodecanoic acid. The specific activity is 18415.400 pmol/min/µg, as measured under the described conditions.

Materials
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)
ASAH2 Protein, Mouse (HEK293, His) (HY-P76735)
Substrate: C12-ceramide (Avanti Polar Lipids, 50 mM stock in Chloroform)
Standard: Sphingosine
o-Phthaldialdehyde (o-PA): 50 mg/mL stock in DMSO
F16 Black Maxisorp Plate

Procedure
1. Standard Curve: Dilute Sphingosine (standard) in sssay buffer to concentrations of 0, 1.5625, 3.125, 6.25, 12.5, 25, 50 μmol/L. Add 200 μL of each concentration to the wells of a black microplate. Read the fluorescence with excitation and emission wavelengths of 330 nm and 450 nm (top read). Plot the measured RFU on the y-axis and the concentration of the standard (μmol/L) on the x-axis to create the standard curve and obtain the standard curve equation.
2. Dissolve 10 μL of 50 mM substrate in 1.99 mL of assay buffer to achieve a concentration of 250 μM.
3. Dilute Mouse ASAH2 to 0.05 μg/mL in assay buffer.
4. Mix 200 μL of 250 μM substrate with 50 μL of 0.05 μg/mL Mouse ASAH2. Include one blank with 50 μL of Mouse ASAH2 and 200 μL of assay buffer, and another blank with 200 μL of substrate and 50 μL of assay buffer.
5. Incubate at 37°C for 1 hour, then heat at 95-100°C for 5 minutes to stop the reaction.
6. Dilute o-PA to 2 mg/mL in o-PA buffer.
7. Add 250 μL of o-PA mixture to all reactions and mix well.
8. Incubate at room temperature for 10 minutes.
9. Add 200 μL (in duplicate) of the reaction mixture and controls to the plate.
10. Read the fluorescence in endpoint mode at excitation and emission wavelengths of 330 nm and 450 nm, respectively. 11. Calculate specific activity:

Mouse

HEK293

N-His

Q9JHE3/NP_061300.1 (T34-T756)

54447  [NCBI]

Lumenal Domain

TSGTIENHKDSGNHWFSTTLGSTTTQPPPITQTPNFPSFRNFSGYYIGVGRADCTGQVSDINLMGYGKNGQNARGLLTRLFSRAFILADPDGSNRMAFVSVELCMISQRLRLEVLKRLESKYGSLYRRDNVILSAIHTHSGPAGFFQYTLYILASEGFSNRTFQYIVSGIMKSIDIAHTNLKPGKIFINKGNVANVQINRSPSSYLLNPQSERARYSSNTDKEMLVLKLVDLNGEDLGLISWFAIHPVSMNNSNHFVNSDNMGYAAYLFEQEKNKGYLPGQGPFVAGFASSNLGDVSPNILGPHCVNTGESCDNDKSTCPNGGPSMCMASGPGQDMFESTHIIGRIIYQKAKELYASASQEVTGPVLAAHQWVNMTDVSVQLNATHTVKTCKPALGYSFAAGTIDGVSGLNITQGTTEGDPFWDTLRDQLLGKPSEEIVECQKPKPILLHSGELTIPHPWQPDIVDVQIVTVGSLAIAAIPGELTTMSGRRFREAIKKEFALYGMKDMTVVIAGLSNVYTHYITTYEEYQAQRYEAASTIYGPHTLSAYIQLFRDLAKAIATDTVANMSSGPEPPFFKNLIASLIPNIADRAPIGKHFGDVLQPAKPEYRVGEVVEVIFVGANPKNSAENQTHQTFLTVEKYEDSVADWQIMYNDASWETRFYWHKGILGLSNATIYWHIPDTAYPGIYRIRYFGHNRKQELLKPAVILAFEGISSPFEVVTT

105-115 kDa

Greater than 95% as determined by reducing SDS-PAGE

Lyophilized powder.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.