Recombinant Human Cystatin F Protein, CF Summary
Product Specifications
Gly20-His145, with a C-terminal 6-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.
1889-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 7.5. |
| Shipping | The product is shipped at ambient temperature. 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
- Assay Buffer: 50 mM MES, pH 6.0
- Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
- Recombinant Human Cystatin F (rhCystatin F) (Catalog # 1889-PI)
- Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
- Substrate: Z-Leu-Arg-AMC (Catalog # ES008), 10 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 Assay Buffer with 5 mM DTT.
- 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 F (MW: 15,356 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 3.9 nM.
- Gently mix equal volumes of the rhCystatin F 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 five-fold dilution of reaction mixture with Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the incubated mixtures into a black well 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 50% inhibition concentration (IC50) value for rhCystatin F by plotting RFU/min (or specific activity) versus 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 F curve: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.20 nM
- Substrate: 10 µM
Reconstitution Calculator
Background: Cystatin F
Cystatin F, also known as leukocystatin and CMAP (Cystatin‑like Metastasis‑Associated Protein), is a 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 intra disulfide bonds, cystatin F has two extra Cys residues that may be involved in inter disulfide bonds. Indeed, rhCystatin F showed 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).
- 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.
Citations for Recombinant Human Cystatin F 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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Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification.
Authors: Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh GJ, Yamamoto K, Nishi K, Watts C, Reinheckel T, Schalkwijk J, Zeeuwen PL
J. Biol. Chem., 2006-03-24;281(23):15893-9.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay -
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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