Recombinant Human Cystatin A Protein, CF

Catalog # Availability Size / Price Qty
1407-PI-050
R&D Systems Recombinant Proteins and Enzymes
1 Image
Product Details
Citations (5)
FAQs
Supplemental Products
Reviews

Recombinant Human Cystatin A Protein, CF Summary

Product Specifications

Purity
>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.
Activity
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
Analysis
Met
Predicted Molecular Mass
12 kDa
SDS-PAGE
12 kDa, reducing conditions

Product Datasheets

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.

1407-PI

Formulation Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Reconstitution Reconstitute at 100 μg/mL in sterile 25 mM Tris and 100 mM NaCl, pH 7.5.
Shipping The product is shipped at ambient temperature. 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

Materials
  • 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
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 A

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).

References
  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.
Entrez Gene IDs
1475 (Human)
Alternate Names
AREI; CSTA; cystatin A (stefin A); Cystatin A; cystatin AS; cystatin-A; Stefin A; STF1; STF1Cystatin-AS; STFA; STFAStefin-A

Citations for Recombinant Human Cystatin A 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.

5 Citations: Showing 1 - 5
Filter your results:

Filter by:

  1. 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.
    Authors: Chang KP, Wu CC, Chen HC
    Proteomics, 2010;10(14):2644-60.
    Species: Human
    Sample Types: Whole Cells
    Applications: Bioassay
  2. Cystatin B as a tissue and urinary biomarker of bladder cancer recurrence and disease progression.
    Authors: Feldman AS, Banyard J, Wu CL, McDougal WS, Zetter BR
    Clin. Cancer Res., 2009;15(3):1024-31.
    Species: N/A
    Sample Types: N/A
    Applications: Western Blot
  3. VEGF-A induces angiogenesis by perturbing the cathepsin-cysteine protease inhibitor balance in venules, causing basement membrane degradation and mother vessel formation.
    Authors: Chang SH, Kanasaki K, Gocheva V, Blum G, Harper J, Moses MA, Shih SC, Nagy JA, Joyce J, Bogyo M, Kalluri R, Dvorak HF
    Cancer Res., 2009;69(10):4537-44.
    Species: Mouse
    Sample Types: In Vivo
    Applications: In Vivo
  4. 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
  5. 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

FAQs

No product specific FAQs exist for this product, however you may

View all Proteins and Enzyme FAQs

Fluorogenic Peptide Substrates

Reviews for Recombinant Human Cystatin A Protein, CF

There are currently no reviews for this product. Be the first to review Recombinant Human Cystatin A Protein, CF and earn rewards!

Have you used Recombinant Human Cystatin A Protein, CF?

Submit a review and receive an Amazon gift card.

$25/€18/£15/$25CAN/¥75 Yuan/¥1250 Yen for a review with an image

$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image

Submit a Review