Recombinant Human Cystatin D Protein, CF

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R&D Systems Recombinant Proteins and Enzymes
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Recombinant Human Cystatin D Protein, CF Summary

Product Specifications

>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.
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.
Mouse myeloma cell line, NS0-derived human Cystatin D protein
Thr29-Val142, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Predicted Molecular Mass
15 kDa
15 kDa, reducing conditions

Product Datasheets

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Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.


Formulation Lyophilized from a 0.2 μm filtered solution in MES and NaCl.
Reconstitution Reconstitute at 100 μg/mL in sterile 50 mM Tris, pH 7.0.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

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
Reconstitution Calculator

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.


Background: Cystatin D

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.
Entrez Gene IDs
1473 (Human)
Alternate Names
CST5; CSTD; cystatin 5; Cystatin D; Cystatin-5; cystatin-D; cysteine-proteinase inhibitor; MGC71922

Citations for Recombinant Human Cystatin D 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.

3 Citations: Showing 1 - 3
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  1. 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;281(23):15893-9.
    Species: Human
    Sample Types: Recombinant Protein
    Applications: Enzyme Assay
  2. Characterization of cathepsin L secreted by Sf21 insect cells.
    Authors: Johnson GD, Jiang W
    Arch. Biochem. Biophys., 2005;444(1):7-14.
    Species: N/A
    Sample Types: Protein
    Applications: Enzyme Assay
  3. Murine monoclonal antibody which can distinguish cystatins SA1 and SA2.
    Authors: Ito T, Komiya-Ito A, Okuda K, Minaguchi K, Saitoh E, Yamada S, Kato T
    Mol. Immunol., 2005;42(10):1259-63.
    Species: N/A
    Sample Types: N/A
    Applications: ELISA (Standard)


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Fluorogenic Peptide Substrates

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