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). Cystatin B-deficient mice have increased expression of proteolysis, apoptosis and glial activation genes, which is consistent with the pathology found in the mouse model of human progressive myoclonus epilepsy (EPM1) (3). The mouse Cystatin B consists of 98 amino acid residues (4).
Recombinant Mouse Cystatin B Protein, CF
R&D Systems | Catalog # 1409-PI
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Key Product Details
- R&D Systems E. coli-derived Recombinant Mouse Cystatin B Protein (1409-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
Structure / Form
Monomer and a disulfide-linked multimer
Applications
Inhibition Activity
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Product Specifications
Source
E. coli-derived mouse Cystatin B protein
Met1-Phe98, with an N-terminal Met and 6-His tag
Met1-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 (monomer)
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
1409-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.
- Lieuallen, K. et al. (2001) Hum. Mol. Genet. 10:1867.
- Pennacchio, L.A. and R.M. Myers (1996) Genome Res. 6:1103.
Alternate Names
CSTB, Stefin B
Gene Symbol
CSTB
UniProt
Additional Cystatin B Products
Product Documents for Recombinant Mouse 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 Mouse Cystatin B Protein, CF
For research use only
Related Research Areas
Citations for Recombinant Mouse Cystatin B Protein, CF
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Protocols
View specific protocols for Recombinant Mouse Cystatin B Protein, CF (1409-PI):
Materials
- Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
- Assay Buffer: 50 mM Tris, pH 7.0
- Recombinant Mouse Cystatin B (rmCystatin B) (Catalog # 1409-PI)
- Papain (Sigma, Catalog # P4762)
- 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
- Dilute Papain to 100 µg/mL in Activation Buffer.
- Incubate at room temperature for 15 minutes.
- Prepare a curve of rmCystatin B (MW: 11,991 Da) in Assay Buffer. Make the following serial dilutions: 800, 400, 200, 150, 100, 50, 25 and 12.5 nM.
- Dilute activated Papain to 2.4 µg/mL in Assay Buffer.
- Combine 20 µL of 2.4 µg/mL Papain with 20 µL of rmCystatin B curve dilutions. Include two controls containing 20 µL Assay Buffer with 20 µL of 2.4 µg/mL Papain.
- Incubate at room temperature for 10 minutes.
- After incubation, dilute the rmCystatin B curve 5-fold by adding 160 µL of Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- In a plate, load 50 µL of the diluted rmCystatin B curve, and the start the reaction by adding 50 µL of 200 µM Substrate to wells.
- Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
- Derive the 50% inhibition concentration (IC50) value for rmCystatin 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: 0.012 µg
- Substrate: 100 µM
- rmCystatin B curve: 40, 20, 10, 7.5, 5, 2.5, 1.25 and 0.625 nM