Recombinant Human Cystatin B Protein, CF Summary
Product Specifications
Met2-Phe98, with an N-terminal Met and 7-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
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.
|
Assay Procedure
- 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).
- 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
Reconstitution Calculator
Background: Cystatin B
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).
- 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.
Citations for Recombinant Human Cystatin B Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
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Citations: Showing 1 - 4
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Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidic dPCR chip
Authors: J Hu, T Gou, W Wu, J Sun, S Zhang, S Zhou, J Yin, Y Mu
Anal. Chim. Acta, 2019-05-17;1076(0):118-124.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Regulation of TGF-beta1-driven differentiation of human lung fibroblasts: emerging roles of cathepsin B and cystatin C.
Authors: Kasabova M, Joulin-Giet A, Lecaille F, Gilmore B, Marchand-Adam S, Saidi A, Lalmanach G
J Biol Chem, 2014-04-30;289(23):16239-51.
Species: Human
Sample Types:
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Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification.
Authors: Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh GJ, Yamamoto K, Nishi K, Watts C, Reinheckel T, Schalkwijk J, Zeeuwen PL
J. Biol. Chem., 2006-03-24;281(23):15893-9.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay -
Characterization of cathepsin L secreted by Sf21 insect cells.
Authors: Johnson GD, Jiang W
Arch. Biochem. Biophys., 2005-10-21;444(1):7-14.
Applications: Enzyme Assay
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