Recombinant Human SMPD1 Protein, CF Summary
His62-Pro628, 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 Tris and NaCl.|
|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: 50 mM MES, 0.5 µM ZnCl2, pH 6.5
- Developing Buffer: 0.2 M NaOH
- Recombinant Human SMPD1 (rhSMPD1) (Catalog # 5348-PD)
- Substrate: 2-N-Hexadecanoyl-4-nitrophenylphosphorylcholine (HNPPC) (Gojira Fine Chemicals, Catalog # HN1004), 50 mM stock solution in methanol. Note: Incubate at 37 °C for 5 minutes and vortex to dissolve.
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhSMPD1 to 2 µg/mL in Assay Buffer and incubate at 37 °C for 10 minutes.
- Dilute Substrate to 1 mM in Assay Buffer and incubate at 37 °C for 10 minutes.
- Combine 50 µL of rhSMPD1 and 50 µL of Substrate in a plate. Create Substrate Blanks by using Assay Buffer in place of rhSMPD1.
- Cover plate and incubate at room temperature for 20 minutes in the dark.
- Stop the reaction by adding 100 µL of Developing Buffer to each well.
- Read (top read) absorbance in endpoint mode at 410 nm.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Adjusted Abs* (OD) x Conversion Factor** (pmol/OD)|
|Incubation time (min) x amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard p-Nitrophenol (Sigma-Aldrich, Catalog # 241326).Per Well:
- rhSMPD1: 0.10 µg
- Substrate: 250 µM
Sphingomyelin phosphodiesterase, also known as acid sphingomyelinase and encoded by the SMPD1 gene, is a lysosomal phosphodiesterase which belongs to the acid sphingomyelinase family (1). SMPD1 catalyzes the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Ceramide, a bioactive lipid, has emerged as an important signaling molecule involved in a variety of cellular processes such as cell differentiation, apoptosis, and proliferation (2). Activation of SMPD1 occurs by the removal, chemical modification or dimerization of its C-terminal cysteine residue (3). Deficiencies of SMPD1 result in a lysosomal storage disorder referred to as Niemann-Pick disease (4). Recombinant human SMPD1 was expressed without the last three C-terminal residues, and is therefore constitutively active.
- Schuchman, E.H. et al. (1991) J. Biol. Chem. 266:8531.
- Melendez, A.J. et al. (2008) Biochim. Biophys. Acta 1784:66.
- Qiu, H. et al. (2003) J. Biol. Chem. 278:32744.
- Smith, E.L. and Schuchman, E.H. (2008) FASEB J. 22:3419.
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