Recombinant Human Cystatin E/M Protein, CF Summary
Product Specifications
Arg29-Met149, 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.
1286-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
- Assay Buffer: 50 mM Tris, pH 7.0
- Dithiothreitol (DTT) (Sigma, Catalog # D0632)
- Recombinant Human Cystatin E/M (rhCystatin E/M) (Catalog # 1286-PI)
- Papain (Sigma, Catalog # P-4762)
- 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 the Assay Buffer on ice.
- Dilute Papain to 100 µg/mL in Assay Buffer with 5 mM DTT.
- Incubate at room temperature for 15 minutes.
- After incubation, dilute activated Papain to 2.05 ng/µL in Assay Buffer.
- Prepare a curve of rhCystatin E/M (MW: 14,953Da) in Assay Buffer. Make the following serial dilutions: 2000, 500, 250, 125, 62.5, 31.25, 15.625, 5, and 1 nM.
- Combine equal volumes of the rhCystatin E/M curve dilutions and 2.05 ng/µL active Papain. Include a control (in duplicate) containing equal volumes of Assay Buffer and 2.05 ng/µL Papain without adding any rhCystatin E/M.
- Incubate mixtures at 37 °C for 10 minutes.
- Dilute the reaction mixture 5 fold with Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of the incubated mixtures in a plate, and start the reaction by adding 50 µL of 200 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) for rhCystatin E/M by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity of Papain 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).
- Papain: 0.01025 µg
- rhCystatin E/M curve: 100, 25, 12.5, 6.25, 3.125, 1.5625, 0.781, 0.25, and 0.05 nM
- Substrate: 100 µM
Reconstitution Calculator
Background: Cystatin E/M
Cystatin E/M encoded by the CST6 gene is a member of family 2 of the cystatin superfamily (1, 2). It inhibits papain and cathepsin B, two of the cysteine proteases. Its mRNA was found in many tissues by the two groups who did initial cloning (1, 2). However, its protein was found only in skin and sweat glands by a third group (3). In addition to being a cysteine protease inhibitor, cystatin E/M is also a substrate for transglutaminases (3). It is required for viability and for correct formation of cornified layers in the epidermis and hair follicles, as ichq mice, with a null mutation in the cystatin E/M gene, have defects in epidermal cornification and die between 5 and 12 days of age (4). Cystatin E/M expression and function may not be limited to cutaneous epithelia. For example, it is found in rat brain and is induced during neuronal cell differentiation (5).
- Sotiropoulou, G. et al. (1997) J. Biol. Chem. 272:903.
- Ni, J. et al. (1997) J. Biol. Chem. 272:10853.
- Zeeuwen, P.L. et al. (2001) J. Invest. Dermatol. 116:693.
- Zeeuwen, P.L. et al. (2002) Hum. Mol. Genet. 11:2867.
- Hong, J. et al. (2002) J. Neurochem. 81:922.
Citations for Recombinant Human Cystatin E/M 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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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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Average Rating: 5 (Based on 1 Review)
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Reason for Rating: Using the cst6 protein as a positive control for western blot. It works well.
