Recombinant Human Cystatin SN Protein, CF Summary
Product Specifications
Trp21-Ser141, 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.
1285-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in MES and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 25 mM MES, 150 mM NaCl, pH 6.5. |
| 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 SN (rhCystatin SN) (Catalog # 1285-PI)
- Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
- Substrate: Z-Leu-Arg-AMC (Catalog # ES008)
- 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 15 minutes on ice.
- Dilute rhCathepsin L to 0.5 µg/mL in Activation Buffer.
- Prepare a curve of rhCystatin SN (MW: 15,677 Da) in Assay Buffer. Make the following serial dilutions: 2000, 500, 200, 100, 66.7, 44.4, 22.2, 11.1, and 2.78 nM.
- Gently mix equal volumes of the rhCystatin SN 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 ºC for 15 minutes.
- After incubation, perform a 1/5 dilution of each reaction mixture with Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load into a black well plate 50 µL of the incubated mixtures, and start the reaction by adding 50 µL of 20 µM 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% inhibition concentration (IC50) value for rhCystatin SN 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: 0.0025 µg
- rhCystatin SN curve: 100, 25, 10, 5, 3.33, 2.22, 1.11, 0.555, and 0.139 nM
- Substrate: 10 µM
Reconstitution Calculator
Background: Cystatin SN
Cystatin SN is a member of family 2 of the cystatin superfamily (1). Together with cystatins S and SA, it is produced by the salivary gland and secreted largely in the submandibular/sublingual saliva (2). Cystatin SN inhibits members of the papain family including cathepsins B, C, H and L (3).
- Abrahamson, M. (1994) Methods Enzymol. 244:685.
- Baron, A.C. et al. (1999) Oral Dis. 5:344.
- Baron, A. et al. (1999) Oral Dis. 5:234.
Citation for Recombinant Human Cystatin SN 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.
1 Citation: Showing 1 - 1
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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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