Recombinant Human Cystatin B Protein, CF

• Reactivity: Hu
• Applications: Inhibition Activity
• Format: Carrier-Free

Recombinant Human Cystatin B Protein, CF Summary

• Details of Functionality: 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.
• Source: E. coli-derived human Cystatin B protein
  Met2-Phe98, with an N-terminal Met and 7-His tag
• Accession #: P04080
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CSTB
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Inhibition Activity
• Theoretical MW: 12 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 13 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute at 100 μg/mL in sterile 25 mM Tris and 100 mM NaCl, pH 7.5.
• 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
  1. Chill Activation Buffer on ice.
  2. Dilute Papain to 100 μg/mL in Activation Buffer.
  3. Incubate at room temperature for 15 minutes.
  4. 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.
  5. Dilute activated Papain to 2.4 μg/mL in Assay Buffer.
  6. 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.
  7. Incubate mixtures at room temperature for 15 minutes.
  8. Dilute Substrate to 200 μM in assay Buffer.
  9. Perform a five fold dilution with Assay Buffer to the incubated mixture of rhCystatin B and Papain.
  10. Load 50 μL of diluted incubated mixture into a plate and start the reaction by adding 50 μL of 200 μM substrate.
  11. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
  12. Derive the 50% inhibiting concentration (IC₅₀) value for rhCystatin B by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
  13. 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Cystatin B Protein, CF

• CPI-B
• CST6cystatin B (liver thiol proteinase inhibitor)10STFBcystatin-B
• CSTB
• cystatin B (stefin B)
• Cystatin B
• EPM1
• Liver thiol proteinase inhibitor
• PME
• Stefin B
• stefin-B

Background

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).

1. Abrahamson, M. (1994) Methods Enzymol. 244:685.
2. Pol, E. and I. Bjork (1999) Biochemistry 38:10519.
3. Pennacchio, L.A. et al. (1996) Science 271:1731.
4. Zhang, R. et al. (2003) Glia 42:194.
5. Zerovnik, E. et al. (2002) Biochim. Biophys. Acta 1594:1.

Publications for Cystatin B/Stefin B (1408-PI)(4)

Publications using 1408-PI

• J Hu, T Gou, W Wu, J Sun, S Zhang, S Zhou, J Yin, Y Mu Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidic dPCR chip Anal. Chim. Acta, 2019-05-17;1076(0):118-124. 2019-05-17 [PMID: 31203955] (Bioassay, Human)
• Kasabova M, Joulin-Giet A, Lecaille F, Gilmore B, Marchand-Adam S, Saidi A, Lalmanach G Regulation of TGF-beta1-driven differentiation of human lung fibroblasts: emerging roles of cathepsin B and cystatin C. J Biol Chem, 2014-04-30;289(23):16239-51. 2014-04-30 [PMID: 24790080] (Human)
• Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh GJ, Yamamoto K, Nishi K, Watts C, Reinheckel T, Schalkwijk J, Zeeuwen PL 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. J. Biol. Chem., 2006-03-24;281(23):15893-9. 2006-03-24 [PMID: 16565075] (Enzyme Assay, Human)
• Johnson GD, Jiang W Characterization of cathepsin L secreted by Sf21 insect cells. Arch. Biochem. Biophys., 2005-10-21;444(1):7-14. 2005-10-21 [PMID: 16274658] (Enzyme Assay)

Bioinformatics

• Gene Symbol: CSTB
• Uniprot:
  • P04080()