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Recombinant Human Cystatin A Protein, CF

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Product Details

Summary
Reactivity HuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

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Recombinant Human Cystatin A 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 <3.4 nM, as measured under the described conditions.
Source
E. coli-derived human Cystatin A protein
Ile2-Phe98, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
Met
Protein/Peptide Type
Recombinant Enzymes
Gene
CSTA
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
12 kDa, reducing conditions
Publications
Read Publications using
1407-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 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 A (rhCystatin A) (Catalog # 1407-PI)
  • Papain (Sigma, Catalog # P4762)
  • Substrate: N-Carbobenzyloxy-Phe-Arg-7-amido-4-methylcoumarin (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 A (MW: 11,822 Da) in Assay Buffer. Make the following serial dilutions: 400, 200, 100, 66.7, 44.4, 29.6, 19.8, and 6.58 nM.
  5. Dilute activated papain to 2.33 µg/mL in Assay Buffer.
  6. Mix equal volumes of the rhCystatin A curve dilutions and diluted activated Papain. Include a control (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 A and Papain.
  10. Load 50 µL of diluted incubated mixture into a black well 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 value (IC50) for rhCystatin A 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: 0.01165 µg
  • Substrate: 100 µM
  • rhCystatin A curve: 20, 10, 5, 3.34, 2.22, 1.48, 0.99, and 0.329 nM

Notes

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

Alternate Names for Recombinant Human Cystatin A Protein, CF

  • AREI
  • CSTA
  • cystatin A (stefin A)
  • Cystatin A
  • cystatin AS
  • cystatin-A
  • Stefin A
  • STF1
  • STF1Cystatin-AS
  • STFA
  • STFAStefin-A

Background

Cystatin A, also called stefin A, Cystatin AS or keratolinin, is a member of family 1 of the cystatin superfamily (1, 2). Like Cystatin B, it is an intracellular inhibitor regulating the activities of cysteine proteases of the papain family such as cathepsins B, H and L (3). For example, immunohistochemical analysis of Cystatin A and cathepsin L is a useful indicator for malignancy in human epidermal keratinocytes (4). The ratio of cathepsin B and Cystatin A can be used in the differential diagnosis and treatment of patients with prostate carcinoma (5). The human Cystatin A consists of 98 amino acids (1).

  1. Kartasova, T. et al. (1987) Nucleic Acids Res. 15:5945.
  2. Abrahamson, M. (1994) Methods Enzymol. 244:685.
  3. Jenko, S. et al. (2003) J. Mol. Biol. 326:875.
  4. Palungwachira, P. et al. (2002) J. Dermatol. 29:573.
  5. Sinha, A.A. et al. (2002) Cancer 94:3141.

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Publications for Cystatin A (1407-PI)(5)

We have publications tested in 2 confirmed species: Human, Mouse.

We have publications tested in 5 applications: Bioassay, ELISA (Standard), Enzyme Assay, In Vivo, Western Blot.


Filter By Application
Bioassay
(1)
ELISA (Standard)
(1)
Enzyme Assay
(2)
In Vivo
(1)
Western Blot
(1)
All Applications
Filter By Species
Human
(2)
Mouse
(1)
All Species
Showing Publications 1 - 5 of 5.
Publications using 1407-PI Applications Species
Chang KP, Wu CC, Chen HC Identification of candidate nasopharyngeal carcinoma serum biomarkers by cancer cell secretome and tissue transcriptome analysis: potential usage of cystatin A for predicting nodal stage and poor prognosis. Proteomics, 2010-07-01;10(14):2644-60. 2010-07-01 [PMID: 20461718] (Bioassay, ELISA (Standard), Human) Bioassay, ELISA (Standard) Human
Chang SH, Kanasaki K, Gocheva V, Blum G, Harper J, Moses MA, Shih SC, Nagy JA, Joyce J, Bogyo M, Kalluri R, Dvorak HF VEGF-A induces angiogenesis by perturbing the cathepsin-cysteine protease inhibitor balance in venules, causing basement membrane degradation and mother vessel formation. Cancer Res., 2009-05-12;69(10):4537-44. 2009-05-12 [PMID: 19435903] (In Vivo, Mouse) In Vivo Mouse
Feldman AS, Banyard J, Wu CL, McDougal WS, Zetter BR Cystatin B as a tissue and urinary biomarker of bladder cancer recurrence and disease progression. Clin. Cancer Res., 2009-02-01;15(3):1024-31. 2009-02-01 [PMID: 19188175] (Western Blot) Western Blot
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) 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) Enzyme Assay

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Bioinformatics

Gene Symbol CSTA
Uniprot