Cystatin A, also called stefin A, Cystatin AS or keratolinin, is a member of family 1 of the cystatin superfamily (1, 2). Like Cystatin B, it is an intracellular inhibitor regulating the activities of cysteine proteases of the papain family such as cathepsins B, H and L (3). For example, immunohistochemical analysis of Cystatin A and cathepsin L is a useful indicator for malignancy in human epidermal keratinocytes (4). The ratio of cathepsin B and Cystatin A can be used in the differential diagnosis and treatment of patients with prostate carcinoma (5). The human Cystatin A consists of 98 amino acids (1).
Recombinant Human Cystatin A Protein, CF
R&D Systems | Catalog # 1407-PI
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Key Product Details
- R&D Systems E. coli-derived Recombinant Human Cystatin A Protein (1407-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
E. coli
Accession Number
Applications
Inhibition Activity
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Product Specifications
Source
E. coli-derived human Cystatin A protein
Ile2-Phe98, with an N-terminal Met and 6-His tag
Ile2-Phe98, with an N-terminal Met and 6-His tag
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
N-terminal Sequence Analysis
Met
Predicted Molecular Mass
12 kDa
SDS-PAGE
12 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 <3.4 nM, as measured under the described conditions.
The IC50 value is <3.4 nM, as measured under the described conditions.
Formulation, Preparation, and Storage
1407-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 100 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.
|
Calculators
Background: Cystatin A
References
- Kartasova, T. et al. (1987) Nucleic Acids Res. 15:5945.
- Abrahamson, M. (1994) Methods Enzymol. 244:685.
- Jenko, S. et al. (2003) J. Mol. Biol. 326:875.
- Palungwachira, P. et al. (2002) J. Dermatol. 29:573.
- Sinha, A.A. et al. (2002) Cancer 94:3141.
Alternate Names
AREI, CSTA, Stefin A, STF1, STFA
Entrez Gene IDs
1475 (Human)
Gene Symbol
CSTA
UniProt
Additional Cystatin A Products
Product Documents for Recombinant Human Cystatin A Protein, CF
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Recombinant Human Cystatin A Protein, CF
For research use only
Related Research Areas
Citations for Recombinant Human Cystatin A Protein, CF
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Protocols
View specific protocols for Recombinant Human Cystatin A Protein, CF (1407-PI):
Materials
- Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
- Assay Buffer: 50 mM Tris, pH 7.0
- Recombinant Human Cystatin A (rhCystatin A) (Catalog # 1407-PI)
- Papain (Sigma, Catalog # P4762)
- Substrate: N-Carbobenzyloxy-Phe-Arg-7-amido-4-methylcoumarin (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 A (MW: 11,822 Da) in Assay Buffer. Make the following serial dilutions: 400, 200, 100, 66.7, 44.4, 29.6, 19.8, and 6.58 nM.
- Dilute activated papain to 2.33 µg/mL in Assay Buffer.
- Mix equal volumes of the rhCystatin A curve dilutions and diluted activated Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
- Incubate mixtures at room temperature 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 A and Papain.
- Load 50 µL of diluted incubated mixture into a black well 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, respectively, for 5 minutes in kinetic mode.
- Derive the 50% inhibiting value (IC50) for rhCystatin A 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 (AMC) (Sigma, Catalog # A-9891).
Per Well:
- Papain: 0.01165 µg
- Substrate: 100 µM
- rhCystatin A curve: 20, 10, 5, 3.34, 2.22, 1.48, 0.99, and 0.329 nM
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