Recombinant Human Cystatin D Protein, CF Summary
Product Specifications
Thr29-Val142, 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.
1202-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in MES and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 50 mM Tris, pH 7.0. |
| 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 Tris, 5 mM DTT, pH 7.0
- Assay Buffer: 50 mM Tris, pH 7.0
- Recombinant Human Cystatin D (rhCystatin D) (Catalog # 1202-PI)
- Papain (Sigma, Catalog # P4762)
- Substrate: Z-Phe-Arg-AMC (Catalog # ES009), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Chill Activation Buffer on ice.
- Dilute Papain to 100 µg/mL in Activation Buffer.
- Incubate at room temperature for 15 minutes.
- Prepare a dilution curve of rhCystatin D (MW: 15,273 Da) in Assay Buffer. Make the following serial dilutions: 6000, 3000, 1000, 500, 250, 100, 50, 10, and 1 nM.
- Dilute activated Papain to 2 µg/mL in Activation Buffer.
- Mix equal volumes of the rhCystatin D curve dilutions and the diluted active Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
- Incubate mixtures at 37 °C for 15 minutes.
- Dilute Substrate to 200 µM in Assay Buffer.
- Perform a five-fold dilution with Assay Buffer to the incubated mixture of rhCystatin D curve and Papain.
- Load 50 µL of diluted incubated mixture into a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank by combining 50 μL of 200 μM Substrate and 50 μL Assay Buffer.
- Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
- Derive the 50% inhibition concentration (IC50) for rhCystatin D by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for Papain at each point may be derived 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 (AMC) (Sigma, Catalog # A-9891).
- Papain: 0.010 µg
- Substrate: 100 µM
- rhCystatin D: 300, 150, 50, 25, 12.5, 5, 2.5, 0.5, and 0.05 nM
Reconstitution Calculator
Background: Cystatin D
Cystatin D is a member of family 2 of the cystatin superfamily (1). In contrast to other members of family 2, Cystatin D has restricted tissue distribution and has been found only in saliva and tears. Two allelic variants (Arg46 and Cys46) are known in the human protein and they are not significantly different in their inhibitory activity against papain and cathepsins B, H, L and S (2). Recombinant Human Cystatin D corresponds to the Arg46 variant. The functions of Cystatin D are largely unknown. However, Cystatin D has been shown to inhibit coronavirus replication at its physiological concentration (0.12‑1.9 μM) and has been suggested to play a protective role against proteases present in the oral cavity (3).
- Freije, J.P. et al. (1993) J. Biol. Chem. 268:15737.
- Balbin, M. et al. (1994) J. Biol. Chem. 269:23156.
- Collins, A.R. and A. Grubb (1998) Oral Microbiol. Immunol. 13:59.
Citations for Recombinant Human Cystatin D 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 - 3
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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 -
Murine monoclonal antibody which can distinguish cystatins SA1 and SA2.
Authors: Ito T, Komiya-Ito A, Okuda K, Minaguchi K, Saitoh E, Yamada S, Kato T
Mol. Immunol., 2005-01-08;42(10):1259-63.
Applications: ELISA (Standard)
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