Recombinant Human SMPD1 Protein, CF

Catalog # Availability Size / Price Qty
5348-PD-010
Product Details
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Recombinant Human SMPD1 Protein, CF Summary

Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave 2-N-Hexadecanoylamino-4-nitrophenylphosphorylcholine (HNPPC). The specific activity is >1,700 pmol/min/μg, as measured under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived human SMPD1 protein
His62-Pro628, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Analysis
His62
Predicted Molecular Mass
66 kDa
SDS-PAGE
71, 77, and 85 kDa, reducing conditions

Product Datasheets

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.

5348-PD

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.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  • 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
  1. Dilute rhSMPD1 to 2 µg/mL in Assay Buffer and incubate at 37 °C for 10 minutes.
  2. Dilute Substrate to 1 mM in Assay Buffer and incubate at 37 °C for 10 minutes.
  3. Combine 50 µL of rhSMPD1 and 50 µL of Substrate in a plate. Create Substrate Blanks by using Assay Buffer in place of rhSMPD1.
  4. Cover plate and incubate at room temperature for 20 minutes in the dark.
  5. Stop the reaction by adding 100 µL of Developing Buffer to each well.
  6. Read (top read) absorbance in endpoint mode at 410 nm.
  7. 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
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: SMPD1

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.

References
  1. Schuchman, E.H. et al. (1991) J. Biol. Chem. 266:8531.
  2. Melendez, A.J. et al. (2008) Biochim. Biophys. Acta 1784:66.
  3. Qiu, H. et al. (2003) J. Biol. Chem. 278:32744.
  4. Smith, E.L. and Schuchman, E.H. (2008) FASEB J. 22:3419.
Long Name
Sphingomyelin Phosphodiesterase 1, Acid Lysosomal
Entrez Gene IDs
6609 (Human); 20597 (Mouse); 308909 (Rat)
Alternate Names
Acid sphingomyelinase; aSMase; ASMsphingomyelin phosphodiesterase; EC 3.1.4.12; NPD; SMPD1; sphingomyelin phosphodiesterase 1, acid lysosomal

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Recombinant Human SMPD1 Protein, CF
By Anonymous on 05/23/2016
Application: Enzymatic activity in vitro