Recombinant Human Cystatin SA Protein, CF Summary
Product Specifications
Trp21-Ala141, 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.
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.
|
Assay Procedure
- 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
- Dilute rhCathepsin L to 40 µg/mL in Activation Buffer.
- Incubate on ice for 15 minutes.
- After incubation, dilute activated rhCathepsin L to 0.52 µg/mL in Activation Buffer.
- 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.
- 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.
- Incubate mixtures at 37 °C for 15 minutes.
- Perform 1/5 dilution of reaction mixtures with Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- 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. - Derive the 50% inhibiting concentration (IC50) value for rhCystatin SA by plotting RFU/min (or specific activity vs. concentration with 4-PL fitting.
- 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).
- 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
Reconstitution Calculator
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).
- Abrahamson, M. (1994) Methods Enzymol. 244:685.
- Baron, A.C. et al. (1999) Exp. Oral. Biol. 5:344.
- Baron, A. et al. (1999) Oral Dis. 5:234.
Citations for Recombinant Human Cystatin SA Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 2
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Identification of cystatin SA as a novel inhibitor of acid ceramidase.
Authors: Eliyahu E, Shtraizent N, He X
J. Biol. Chem., 2011-08-16;286(41):35624-33.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Characterization of cathepsin L secreted by Sf21 insect cells.
Authors: Johnson GD, Jiang W
Arch. Biochem. Biophys., 2005-10-21;444(1):7-14.
Applications: Enzyme Assay
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