Recombinant Human Cystatin SA Protein, CF

R&D Systems | Catalog # 1201-PI

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Key Product Details

  • R&D Systems NS0-derived Recombinant Human Cystatin SA Protein (1201-PI)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

NS0

Accession Number

P09228

Applications

Inhibition Activity
View all Cystatin SA Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived human Cystatin SA protein
Trp21-Ala141, with a C-terminal 10-His tag

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.

N-terminal Sequence Analysis

Trp21

Predicted Molecular Mass

16 kDa

SDS-PAGE

16 kDa, reducing conditions

Activity

Measured by its ability to inhibit active Cathepsin L cleavage of a fluorogenic peptide substrate Z-LR-AMC (Catalog # ES008).
The IC50 value is<8.0 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

1201-PI
Formulation Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Reconstitution Reconstitute at 100 μg/mL in sterile 25 mM Tris and 150 mM NaCl, pH 8.0.
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Calculators

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: Cystatin SA

Cystatin SA is a member of family 2 of the cystatin superfamily (1). Together with cystatins S and SN, it is produced by the salivary gland and secreted largely in the submandibular/sublingual saliva (2). Cystatin SA inhibits cathepsin L, but may not inhibit cathepsins B and H (3). The functions of Cystatin SA are largely unknown. However, it has been suggested to play a role in the control of periodontal tissue destruction (3).

References

  1. Abrahamson, M. (1994) Methods Enzymol. 244:685.
  2. Baron, A.C. et al. (1999) Exp. Oral. Biol. 5:344.
  3. Baron, A. et al. (1999) Oral Dis. 5:234.

Alternate Names

CST2, CSTSA

Entrez Gene IDs

1470 (Human)

Gene Symbol

CST2

UniProt

P09228

Additional Cystatin SA Products

Product Documents for Recombinant Human Cystatin SA Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Cystatin SA Protein, CF

For research use only

Related Research Areas

Cysteine Proteases and Regulators

Citations for Recombinant Human Cystatin SA Protein, CF

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Protocols

View specific protocols for Recombinant Human Cystatin SA Protein, CF (1201-PI):

Materials
  • Activation Buffer: 50 mM MES, 5 mM DTT, pH 6.0
  • Assay Buffer: 50 mM MES, pH 6.0
  • Recombinant Human Cystatin SA (rhCystatin SA) (Catalog # 1201-PI)
  • Recombinant Human Cathepsin L (Catalog # 952-CY)
  • Substrate: Z-Leu-Arg-AMC (Catalog # ES008), 2 mM stock in DMSO.
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

  1. Dilute rhCathepsin L to 40 µg/mL in Activation Buffer.
  2. Incubate on ice for 15 minutes.
  3. After incubation, dilute activated rhCathepsin L to 0.52 µg/mL in Activation Buffer.
  4. Prepare a curve of rhCystatin SA (MW: 15,711 Da) in Assay Buffer. Make the following serial dilutions:  2000, 667, 222, 111, 55.5, 27.75, 9.25, and 3.08 nM.
  5. Gently mix equal volumes of the rhCystatin SA curve dilutions and the diluted active rhCathepsin L. Include a control (in duplicate) containing Assay Buffer and the diluted active rhCathepsin L.
  6. Incubate mixtures at 37 °C for 15 minutes.
  7. Perform 1/5 dilution of reaction mixtures with Assay Buffer.
  8. Dilute Substrate to 20 µM in Assay Buffer.
  9. Load 50 µL of the incubated mixtures in a plate, and start the reaction by adding 50 µL of 20µM Substrate.
    Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  10. Derive the 50% inhibiting concentration (IC50) value for rhCystatin SA by plotting RFU/min (or specific activity vs. concentration with 4-PL fitting.
  11. The specific activity for rhCathepsin L at each point may be determined using the following formula (if needed):

     Specific Activity (pmol/min/µg) =

 Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
amount of enzyme (µg)

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:

  • rhCathepsin L: 0.0026 µg
  • rhCystatin SA curve: 100, 33.5, 11.1, 5.55, 2.775, 1.39, 0.464, and 0.154 nM
  • Substrate: 10 µM

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