Recombinant Mouse Arylsulfatase A/ARSA Protein, CF
Recombinant Mouse Arylsulfatase A/ARSA Protein, CF Summary
Product Specifications
Ser18-Ser506, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
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.
2858-SU
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| 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.
|
Assay Procedure
- Assay Buffer: 50 mM Sodium Acetate, pH 5.0
- Recombinant Mouse Arylsulfatase A/ARSA (rmARSA) (Catalog # 2858-SU)
- Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N-7251), 100 mM stock in diH2O
- 0.2 M Sodium Hydroxide
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmARSA to 20 µg/mL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Combine in triplicate 75 µL of 20 µg/mL rmARSA and 75 µL of 2 mM Substrate. Include a Substrate Blank containing Assay Buffer in place of rmARSA.
- Incubate reactions for 30 minutes at 37 °C.
- Stop reactions by adding 150 µL 0.2 M sodium hydroxide to each for a final concentration of 0.1 M sodium hydroxide per reaction.
- Load into a clear 96-well plate 200 µL from each reaction vial.
- Read at 510 nm (absorbance) in endpoint mode.
- 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 4-Nitrocatechol (PNC) (Sigma, Catalog # N15553).
- rmARSA: 1 µg
- Substrate: 0.5 mM
Reconstitution Calculator
Background: Arylsulfatase A/ARSA
As a member of the sulfatase family, arylsulfatase A is encoded by the ARSA gene and required for the lysosomal degradation of cerebroside-3-sulfate, a sphingolipid sulfate ester and a major constituent of the myelin sheet (1). ARSA deficiency results in metachromatic leukodystrophy (MLD), a lysosomal storage disease in the central and peripheral nervous systems with severe and progressive neurological symptoms (2). The deduced amino acid sequence of mouse ARSA consists of a signal peptide (residues 1-17) and a mature chain (residues 18-506) (3). Recombinant mouse ARSA corresponds to the mature chain and has sulfatase activity as described in Activity Assay Protocol.
- Lukatela, G. et al. (1998) Biochemistry 37:3654.
- Parenti, G. et al. J. (1997) Curr. Opin. Genet. & Dev. 7:386.
- Kreysing, J. et al. (1994) Genomics 19:249.
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