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).
Recombinant Human Cystatin E/M Protein, CF
R&D Systems | Catalog # 1286-PI
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Key Product Details
- R&D Systems NS0-derived Recombinant Human Cystatin E/M Protein (1286-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
Applications
Inhibition Activity
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Product Specifications
Source
Mouse myeloma cell line, NS0-derived human Cystatin E/M protein
Arg29-Met149, with a C-terminal 10-His tag
Arg29-Met149, with a C-terminal 10-His tag
Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
Arg29
Predicted Molecular Mass
15 kDa
SDS-PAGE
15 kDa and 21 kDa, reducing conditions
Activity
Measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC (Catalog # ES009).
The IC50 value is approximately 7.0 nM, as measured under the described conditions.
Reviewed Applications
Read 1 review rated 5 using 1286-PI in the following applications:
Formulation, Preparation, and Storage
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.
|
Calculators
Background: Cystatin E/M
References
- 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.
Alternate Names
CST6
Gene Symbol
CST6
UniProt
Additional Cystatin E/M Products
Product Documents for Recombinant Human Cystatin E/M Protein, CF
Certificate of Analysis
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Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Human Cystatin E/M Protein, CF
For research use only
Related Research Areas
Citations for Recombinant Human Cystatin E/M Protein, CF
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Protocols
View specific protocols for Recombinant Human Cystatin E/M Protein, CF (1286-PI):
Materials
- 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).
Per Well:
- 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