Cystatin B, also called stefin B or liver thiol proteinase inhibitor, is a member of family 1 of the cystatin superfamily (1). Like Cystatin A, it is an intracellular inhibitor regulating the activities of cysteine proteases of the papain family such as cathepsins B, H and L (2). Mutations in the Cystatin B gene is the cause of progressive myoclonus epilepsy (EPM1) (3). Because of its expression patterns, Cystatin B can be used as a marker for certain cancers, such as glioblastoma tumors (4). It readily forms amyloid fibrils in vitro (5). The human Cystatin B consists of 98 amino acid residues (3).
Recombinant Human Cystatin B Protein, CF
R&D Systems | Catalog # 1408-PI
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Key Product Details
- R&D Systems E. coli-derived Recombinant Human Cystatin B Protein (1408-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 B protein
Met2-Phe98, with an N-terminal Met and 7-His tag
Met2-Phe98, with an N-terminal Met and 7-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
13 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 <15 nM, as measured under the described conditions.
Formulation, Preparation, and Storage
1408-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 B
References
- Abrahamson, M. (1994) Methods Enzymol. 244:685.
- Pol, E. and I. Bjork (1999) Biochemistry 38:10519.
- Pennacchio, L.A. et al. (1996) Science 271:1731.
- Zhang, R. et al. (2003) Glia 42:194.
- Zerovnik, E. et al. (2002) Biochim. Biophys. Acta 1594:1.
Alternate Names
CSTB, Stefin B
Gene Symbol
CSTB
UniProt
Additional Cystatin B Products
Product Documents for Recombinant Human Cystatin B 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 B Protein, CF
For research use only
Related Research Areas
Citations for Recombinant Human Cystatin B Protein, CF
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Protocols
View specific protocols for Recombinant Human Cystatin B Protein, CF (1408-PI):
Materials
- Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
- Assay Buffer: 50 mM Tris, pH 7.0
- Recombinant Human Cystatin B (rhCystatin B) (Catalog # 1408-PI)
- Papain (Sigma, Catalog # P4762)
- Substrate: N-Carbobenzyloxy-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-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 B (MW: 12,092 Da) in Assay Buffer. Make the following serial dilutions: 4000, 800, 400, 300, 200, 150, 100, 50, 25 and 5 nM.
- Dilute activated Papain to 2.4 μg/mL in Assay Buffer.
- Mix equal volumes of the rhCystatin B curve dilutions and the diluted active Papain. Include a Substrate Blank (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 B and Papain.
- Load 50 μL of diluted incubated mixture into 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, respectively, for 5 minutes in kinetic mode.
- Derive the 50% inhibiting concentration (IC50) value for rhCystatin B 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:.012 μg
- Substrate Z-FR-AMC: 100 μM
- rhCystatin B curve: 200, 40, 20, 15, 10, 7.5, 5, 2.5, 1.25, and 0.25 nM