Recombinant Human Cystatin D Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

Order Details

Recombinant Human Cystatin D 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 <8 nM, under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Cystatin D protein
Thr29-Val142, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Thr29
Protein/Peptide Type
Recombinant Enzymes
Gene
CST5
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

Theoretical MW
15 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
15 kDa, reducing conditions
Publications
Read Publications using
1202-PI in the following applications:

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 MES and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile 50 mM Tris, pH 7.0.
Assay Procedure
  • Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
  • Assay Buffer: 50 mM Tris, pH 7.0
  • Recombinant Human Cystatin D (rhCystatin D) (Catalog # 1202-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

  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 D (MW: 15,273 Da) in Assay Buffer. Make the following serial dilutions: 6000, 3000, 1000, 500, 250, 100, 50, 10, and 1 nM.
  5. Dilute activated Papain to 2 µg/mL in Activation Buffer.
  6. Mix equal volumes of the rhCystatin D curve dilutions and the diluted active Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
  7. Incubate mixtures at 37 °C 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 D curve 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. Include a Substrate Blank by combining 50 μL of 200 μM Substrate and 50 μL Assay Buffer.
  11. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
  12. Derive the 50% inhibition concentration (IC50) for rhCystatin D by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
  13. The specific activity for Papain at each point may be derived 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: 0.010 µg
  • Substrate: 100 µM
  • rhCystatin D: 300, 150, 50, 25, 12.5, 5, 2.5, 0.5, and 0.05 nM

Notes

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

Alternate Names for Recombinant Human Cystatin D Protein, CF

  • CST5
  • CSTD
  • cystatin 5
  • Cystatin D
  • Cystatin-5
  • cystatin-D
  • cysteine-proteinase inhibitor
  • MGC71922

Background

Cystatin D is a member of family 2 of the cystatin superfamily (1). In contrast to other members of family 2, Cystatin D has restricted tissue distribution and has been found only in saliva and tears. Two allelic variants (Arg46 and Cys46) are known in the human protein and they are not significantly different in their inhibitory activity against papain and cathepsins B, H, L and S (2). Recombinant Human Cystatin D corresponds to the Arg46 variant. The functions of Cystatin D are largely unknown. However, Cystatin D has been shown to inhibit coronavirus replication at its physiological concentration (0.12‑1.9 μM) and has been suggested to play a protective role against proteases present in the oral cavity (3).

  1. Freije, J.P. et al. (1993) J. Biol. Chem. 268:15737.
  2. Balbin, M. et al. (1994) J. Biol. Chem. 269:23156.
  3. Collins, A.R. and A. Grubb (1998) Oral Microbiol. Immunol. 13:59.

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Publications for Cystatin D/Cst5 (1202-PI)(3)

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Bioinformatics

Gene Symbol CST5
Entrez
Uniprot