Recombinant Human Cystatin A Protein, CF Summary
Ile2-Phe98, with an N-terminal Met and 6-His tag
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.
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 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.
- 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
- Chill Activation Buffer on ice.
- Dilute Papain to 100 µg/mL in Activation Buffer.
- Incubate at room temperature for 15 minutes.
- 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.
- Dilute activated papain to 2.33 µg/mL in Assay Buffer.
- 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.
- Incubate mixtures at room temperature for 15 minutes.
- Dilute Substrate to 200 µM in Assay Buffer.
- Perform a five-fold dilution with Assay Buffer to the incubated mixture of rhCystatin A and Papain.
- Load 50 µL of diluted incubated mixture into a black well plate, and start the reaction by adding 50 µL of 200 μM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
- Derive the 50% inhibiting value (IC50) for rhCystatin A by plotting RFU/min (or specific activity) vs concentration with 4-PL fitting.
- 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).
- 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
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).
- Kartasova, T. et al. (1987) Nucleic Acids Res. 15:5945.
- Abrahamson, M. (1994) Methods Enzymol. 244:685.
- Jenko, S. et al. (2003) J. Mol. Biol. 326:875.
- Palungwachira, P. et al. (2002) J. Dermatol. 29:573.
- Sinha, A.A. et al. (2002) Cancer 94:3141.
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.
Citations: Showing 1 - 5
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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
Sample Types: Whole Cells
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.
Sample Types: N/A
Applications: Western Blot
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.
Sample Types: In Vivo
Applications: In Vivo
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.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
Characterization of cathepsin L secreted by Sf21 insect cells.
Authors: Johnson GD, Jiang W
Arch. Biochem. Biophys., 2005;444(1):7-14.
Sample Types: Protein
Applications: Enzyme Assay
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Fluorogenic Peptide Substrates
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