Recombinant Human Cystatin C Protein, CF Summary
Met1-Ala146, with a C-terminal 10-His tag
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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 HEPES and NaCl.|
|Reconstitution||Reconstitute at 100 μg/mL in sterile 25 mM HEPES and 150 mM NaCl, pH 7.8.|
|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.
- Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
- Assay Buffer: 50 mM Tris, pH 7.0
- Recombinant Human Cystatin C (rhCystatin C) (Catalog # 1196-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
- 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 C (MW: 14,709 Da) in Assay Buffer. Make the following serial dilutions: 1000 nM, 500 nM, 333 nM, 222 nM, 111 nM, 55.5 nM, 27.8 nM, 13.9 nM.
- Dilute activated Papain to 2 µg/mL in Assay Buffer.
- Mix equal volumes of the rhCystatin C curve dilutions and the diluted active Papain. Include a Papain control (in duplicate) containing Assay Buffer and the diluted active Papain.
- Incubate mixtures at 37 °C for 10 minutes.
- Dilute Substrate to 200 µM in Assay Buffer.
- Perform a five-fold dilution of the incubated mixture of rhCystatin C and Papain with Assay Buffer.
- Load into a black well plate 50 µL of diluted incubated mixture 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 concentration (IC50) for rhCystatin C 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: 0.010 µg
- Substrate: 100 µM
- rhCystatin C: 50 nM, 25 nM, 16.65 nM, 11.1 nM, 5.55 nM, 2.78 nM, 1.39 nM, 0.695 nM
Background: Cystatin C
Cystatin C is a member of family 2 of 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 (Leu68 to Gln) 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).
- Reed, C.H. (2000) British J. Biomed. Sci. 57:323.
- Janowski, R. et al. (2001) Nat. Struct. Biol. 8:316.
- Abrahamson, M. (1994) Methods Enzymol. 244:685.
- Abrahamson, M. et al. (1992) Hum. Genet. 89:377.
- Laterza, O.F. et al. (2002) Clin. Chem. 48:699.
Citations for Recombinant Human Cystatin C 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 - 6
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Extracellular Vesicle Proteins Associated with Systemic Vascular Events Correlate with Heart Failure: An Observational Study in a Dyspnoea Cohort.
Authors: Zhang Y, Vernooij F, Ibrahim I, Ooi S, Gijsberts C, Schoneveld A, Sen K, den Ruijter H, Timmers L, Richards A, Jong C, Mazlan I, Wang J, Lam C, de Kleijn D
PLoS ONE, 2016;11(1):e0148073.
Sample Types: Plasma
Applications: ELISA Developmet
Regulation of TGF-beta1-driven differentiation of human lung fibroblasts: emerging roles of cathepsin B and cystatin C.
Authors: Kasabova M, Joulin-Giet A, Lecaille F, Gilmore B, Marchand-Adam S, Saidi A, Lalmanach G
J Biol Chem, 2014;289(23):16239-51.
Sample Types: Cell Culture Supernates
Applications: ELISA (Standard)
Human cysteine cathepsins are not reliable markers of infection by Pseudomonas aeruginosa in cystic fibrosis.
Authors: Naudin C, Joulin-Giet A, Couetdic G, Plesiat P, Szymanska A, Gorna E, Gauthier F, Kasprzykowski F, Lecaille F, Lalmanach G
PLoS ONE, 2011;6(9):e25577.
Sample Types: Sputum
Applications: Western Blot
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
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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