Cystatin E/M is a member of family 2 of the cystatin superfamily (1‑6). 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 (7). 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 (8). The mouse cystatin E/M has not been characterized and is shown here to inhibit papain. In addition to being a cysteine protease inhibitor, the human cystatin E/M is also a substrate for transglutaminases (9).
Recombinant Mouse Cystatin E/M Protein, CF
R&D Systems | Catalog # 1284-PI
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Key Product Details
- R&D Systems NS0-derived Recombinant Mouse Cystatin E/M Protein (1284-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 mouse Cystatin E/M protein
Glu29-Val149 & Thr35-Val149, both with a C-terminal 10-His tag
Glu29-Val149 & Thr35-Val149, both 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
Glu29 & Thr35
Predicted Molecular Mass
19 kDa
SDS-PAGE
20 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 <7 nM, as measured under the described conditions.
Formulation, Preparation, and Storage
1284-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
- Carninci, P. and Y. Hayashizaki (1999) Meth. Enzymol. 303:19.
- Carninci, P. et al. (2000) Genome Res. 10:1617.
- Shibata, K. et al. (2000) Genome Res. 10:1757.
- Kawai, J. et al. (2001) Nature 409:685.
- 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. (2002) Hum. Mol. Genet. 11:2867.
- Hong, J. et al. (2002) J. Neurochem. 81:922.
- Zeeuwen, P.L. et al. (2001) J. Invest. Dermatol. 116:693.
Alternate Names
CST6
Gene Symbol
CST6
UniProt
Additional Cystatin E/M Products
Product Documents for Recombinant Mouse 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 Mouse Cystatin E/M Protein, CF
For research use only
Related Research Areas
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Protocols
View specific protocols for Recombinant Mouse Cystatin E/M Protein, CF (1284-PI):
Materials
- Assay Buffer: 50 mM Tris, pH 7.0
- Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
- Recombinant Mouse Cystatin E/M (rmCystatin E/M) (Catalog # 1284-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.
- Dilute activated Papain to 2 ng/µL in Assay Buffer.
- Prepare a curve of rmCystatin E/M (MW: 18,954 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 250, 125, 93.75, 62.5, 46.875, 31.25, 15.63, 7.8, and 3.9 nM.
- Combine equal volumes of the rmCystatin E/M curve dilutions and 2 ng/µL active Papain. Include a control (in duplicate) containing equal volumes of Assay Buffer and 2 ng/µL Papain without adding any rmCystatin E/M.
- Incubate mixtures at 37 °C for 15 minutes.
- Make a five-fold dilution of reaction mixture with Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load into a black well plate 50 µL of the incubated mixtures 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 rmCystatin E/M by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for 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: 0.010 µg
- rmCystatin E/M curve: 100, 50, 25, 12.5, 6.25, 4.7, 3.1, 2.3, 1.57, 0.78, 0.39, and 0.195 nM
- Substrate: 100 µM