Cystatin F, also known as leukocystatin and CMAP (Cystatin-like Metastasis-Associated Protein), is a new member of the Cystatin superfamily (1-3). Cystatin F is selectively expressed by hematopoietic cells and may be a biomarker for both liver metastasis and inflammatory lung disorders (3, 4). As a cysteine protease inhibitor, it shows selectivity towards Cathepsin L and legumain (1, 2, 5). Compared to other secreted Cystatins including C, D, E/M, S, SA and SN, which contain two intrachain disulfide bonds, Cystatin F has two extra Cys residues that may be involved in interchain disulfide bonds. rmCystatin F and rhCystatin F (Catalog # http://www.rndsystems.com/product_results.aspx?k=1889-PI">1889‑PI) exhibit disulfide bond-linked dimer formation, which was also the case for an E. coli expressed fusion protein containing mature human Cystatin F and glutathione S-transferase (2).
Recombinant Mouse Cystatin F Protein, CF
R&D Systems | Catalog # 4557-PI
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Key Product Details
- R&D Systems NS0-derived Recombinant Mouse Cystatin F Protein (4557-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
Structure / Form
Disulfide-linked homodimer
Applications
Inhibition Activity
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Product Specifications
Source
Mouse myeloma cell line, NS0-derived mouse Cystatin F protein
Ala19-Gln144, with a C-terminal 6-His tag
Accession # O89098
Ala19-Gln144, with a C-terminal 6-His tag
Accession # O89098
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
Ala19
Predicted Molecular Mass
15 kDa
SDS-PAGE
20 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 <60 nM, as measured under the described conditions.
The IC50 value is <60 nM, as measured under the described conditions.
Formulation, Preparation, and Storage
4557-PI
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Brij-35. |
| 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.
|
Background: Cystatin F
References
- Ni, J. et al. (1998) J. Biol. Chem. 273:24797.
- Halfon, S. et al. (1998) J. Biol. Chem. 273:16400.
- Utsunomiya, T. et al. (2002) Clin. Cancer 8:2591.
- Werle, B. et al. (2003) Biol. Chem. 384:281.
- Gruninger-Leitch, F. et al. (2000) Nat. Biotechnol. 18:66.
Alternate Names
CMAP, CST7, Cystatin 7, Leukocystatin
Entrez Gene IDs
8530 (Human)
Gene Symbol
CST7
UniProt
Additional Cystatin F Products
Product Documents for Recombinant Mouse Cystatin F Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Mouse Cystatin F Protein, CF
For research use only
Related Research Areas
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Protocols
View specific protocols for Recombinant Mouse Cystatin F Protein, CF (4557-PI):
Materials
- Assay Buffer: 50 mM MES, pH 6.0
- Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock
- Recombinant Mouse Cystatin F (rmCystatin F) (Catalog # 4557-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 40 µg/mL in Assay Buffer with 5 mM DTT.
- Incubate on ice for 15 minutes.
- After incubation, dilute activated rhCathepsin L to 0.2 µg/mL in Assay Buffer.
- Prepare a curve of rmCystatin F (MW: 15249 Da) in Assay Buffer. Make the following serial dilutions: 6000, 1000, 500, 250, 125, 62.5, 31.25, 15.625, and 5.208 nM.
- Gently mix equal volumes of the rmCystatin L 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 room temperature for 15 minutes.
- Make a 5 fold dilution of the incubated curve dilutions in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the diluted 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% inhibition concentration (IC50) for rmCystatin F 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: 0.001 µg
- rmCystatin F curve: 300, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.781 and 0.260 nM
- Substrate: 10 µM
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