Recombinant Rat Cystatin C Protein, CF

R&D Systems | Catalog # 6154-PI

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Key Product Details

  • R&D Systems NS0-derived Recombinant Rat Cystatin C Protein (6154-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

NS0

Accession Number

P14841

Applications

Inhibition Activity
View all Cystatin C Proteins and Enzymes »

Product Specifications

Source

Mouse myeloma cell line, NS0-derived rat Cystatin C protein
Met1-Ala140, with a C-terminal 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Gly21

Predicted Molecular Mass

14 kDa

SDS-PAGE

15-25 kDa, reducing conditions

Activity

Measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC (Catalog # ES009).
The IC50 is <15 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

6154-PI
Formulation Supplied as a 0.2 μm filtered solution in HEPES and NaCl.
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 opening.

Background: Cystatin C

Cystatin C is a member of family 2 in the Cystatin superfamily (1). It is involved in processes such as tumor invasion and metastasis, inflammation and some neurological diseases. It inhibits many cysteine proteases such as papain and cathepsins B, H, K, L and S (2, 3). It is ubiquitous in human tissues and body fluids. A point mutation in the gene coding for the 120 amino acid mature Cystatin C causes a hereditary form of amyloid angiopathy in which the protein variant is deposited in the cerebral arteries, leading to fatal cerebral hemorrhage (4). Cystatin C may have additional clinical applications. For example, it is a good marker for glomerular filtration rate (5).

References

  1. Reed, C.H. (2000) British J. Biomed. Sci. 57:323.
  2. Janowski, R. et al. (2001) Nat. Struct. Biol. 8:316.
  3. Abrahamson, M. (1994) Methods Enzymol. 244:685.
  4. Abrahamson, M. et al. (1992) Hum. Genet. 89:377.
  5. Laterza, O.F. et al. (2002) Clin. Chem. 48:699.

Alternate Names

ARMD11, CST3, Gamma-trace, Neuroendocrine basic polypeptide, Post-gamma-globulin

Entrez Gene IDs

1471 (Human); 13010 (Mouse); 25307 (Rat)

Gene Symbol

CST3

UniProt

P14841

Additional Cystatin C Products

Product Documents for Recombinant Rat Cystatin C Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Rat Cystatin C Protein, CF

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

For research use only

Citations for Recombinant Rat Cystatin C Protein, CF

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Protocols

View specific protocols for Recombinant Rat Cystatin C Protein, CF (6154-PI):

Materials
  • Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
  • Assay Buffer: 50 mM Tris, pH 7.0
  • Recombinant Rat Cystatin C (rrCystatin C) (Catalog # 6154-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. Dilute papain to 100 µg/mL in Activation Buffer.
  2. Incubate at room temperature for 15 minutes.
  3. Prepare a dilution curve of rrCystatin C (MW: 14,150 Da) in Assay Buffer. Make the following serial dilutions: 1000, 500, 250, 125, 83.3, 55.6, 37.0, 18.5, 9.26 and 4.63 nM.
  4. Dilute activated papain to 2 µg/mL in Assay Buffer.
  5. Mix equal volumes of the rrCystatin C curve dilutions and the diluted active papain. Include a papain control (in duplicate) containing Assay Buffer and the diluted active papain.
  6. Incubate mixtures at 37 ºC for 15 minutes.
  7. Dilute Substrate to 200 µM in Assay Buffer.
  8. Perform a five-fold dilution of the incubated mixture of rrCystatin C and Papain with Assay Buffer.
  9. Load into plate 50 µL of diluted incubated mixture, and start the reaction by adding 50 µL of 200 µM Substrate.
  10. Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
  11. Derive the 50% inhibition concentration (IC50) value for rrCystatin C by plotting RFU/min vs concentration with 4-PL fitting.
  12. Calculate the specific activity for Papain at each point 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
  • rrCystatin C: 50, 25, 12.5, 6.25, 4.17, 2.78, 1.85, 0.926, 0.463, and 0.2315 nM

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