Measured by its ability to inhibit active Cathepsin L cleavage of a fluorogenic peptide substrate Z-LR-AMC (Catalog # ES008). The IC50 value is <1.0 µM as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Cystatin S protein Ser21-Ala141, with a C-terminal 10-His tag
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Inhibition Activity
Theoretical MW
15.6 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
16 kDa, reducing conditions
Publications
Read Publications using 1296-PI in the following applications:
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.
Buffer
Lyophilized from a 0.2 μm filtered solution in MES and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Reconstitution Instructions
Reconstitute at 1 mg/mL in sterile 25 mM MES, 150 mM NaCl, pH 6.5.
Assay Procedure
Activation Buffer: 50 mM MES, 5 mM DTT, pH 6.0
Assay Buffer: 50 mM MES, pH 6.0
Recombinant Human Cystatin S (rhCystatin S) (Catalog # 1296-PI)
Recombinant Human Cathepsin L (rhCathepsin 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 1 µg/mL in Activation Buffer.
Incubate on ice for 15 minutes.
After incubation, dilute activated rhCathepsin L to 0.5 µg/mL in Assay Buffer.
Prepare a curve of rhCystatin S (MW: 15,550 Da) in Assay Buffer. Make the following serial dilutions: ≥45.7 µM (stock concentration should be ≥ 0.711 mg/mL), 30.5, 20.3, 13.5, 9, 6, 4, and 2.68 µM.
Combine equal volumes of the rhCystatin S 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 a 1/5 dilution of each reaction mixture 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 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% inhibiting concentration (IC50) of rhCystatin S 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).
Per Well:
rhCathepsin L (MW: 25524 Da): 0.0025 µg
rhCystatin S curve: ≥2.286 µM (variable based on stock concentration), 1.525, 1.015, 0.675, 0.45, 0.3, 0.2, and 0.134 µM.
Substrate: 10 µM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Cystatin S Protein, CF
CST4
cystatin 4
Cystatin S
Cystatin-4
cystatin-S
cystatin-SA-III
MGC71923
Salivary acidic protein 1
Background
Cystatin S is a member of family 2 of the cystatin superfamily (1).Like cystatins SA and SN, it is produced by the salivary gland and secreted largely in the submandibular/sublingual saliva (2). Unlike cystatins SA and SN, Cystatin S is not a potent inhibitor of cysteine proteases such as papain and some cathepsins (3). Cystatin S is partially phosphorylated at Ser21 and Ser23 in saliva (4). The functions of Cystatin S are largely unknown. However, it is able to bind more calcium and bind more rapidly to carbonated apatite than cystatins SA or SN, indicating that Cystatin S may be involved in the mineral balance of the tooth (3).
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