Recombinant Mouse Cystatin C Protein, CF Summary
Product Specifications
Met1-Ala140, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
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.
1238-PI
| 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.
|
Assay Procedure
- Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
- Assay Buffer: 50 mM Tris, pH 7.0
- Recombinant Mouse Cystatin C (rmCystatin C) (Catalog # 1238-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 to activate.
- Prepare a dilution curve of rmCystatin C (MW: 14,768 Da) in Assay Buffer. Make the following serial dilutions: 3000 nM, 500 nM, 250 nM, 125 nM, 83.3 nM, 55.6 nM, 37.1 nM, 24.7 nM, and 8.23 nM.
- Dilute activated Papain to 2 µg/mL in Assay Buffer.
- Mix equal volumes of the rmCystatin C curve dilutions and the diluted active Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
- Incubate mixtures at 37 °C for 15 minutes.
- Dilute Substrate to 200 µM in Assay Buffer.
- Perform a five-fold dilution with Assay Buffer to the incubated mixture of rmCystatin C 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 concentration (IC50) of rmCystatin 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).
Per Well:- Papain: 0.01 µg
- Substrate: 100 µM
- rmCystatin C curve: 150, 25, 12.5, 6.25, 4.165, 2.78, 1.885, 1.235, and 0.412 nM
Reconstitution Calculator
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). All mouse tissues analyzed expressed Cystatin C, with relative levels similar to those of rat and human tissues. For all three species, brain and liver had the highest and lowest levels of Cystatin C, respectively, whereas kidney, spleen and muscle had the levels in between (4). The high degree of similarity in distribution and functional properties for mouse, rat and human Cystatin C indicates that a murine model should be relevant for studies of the human disease, hereditary Cystatin C amyloid angiopathy (4).
- 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.
- Hakansson, K. et al. (1996) Comp. Biochem. Physiol. 114B:303.
Citations for Recombinant Mouse 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.
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Citations: Showing 1 - 2
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Urinary clusterin, cystatin C, beta2-microglobulin and total protein as markers to detect drug-induced kidney injury.
Authors: Dieterle F, Perentes E, Cordier A, Roth DR, Verdes P, Grenet O, Pantano S, Moulin P, Wahl D, Mahl A, End P, Staedtler F, Legay F, Carl K, Laurie D, Chibout SD, Vonderscher J, Maurer G
Nat. Biotechnol., 2010-05-01;28(5):463-9.
Applications: ELISA (Standard) -
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., 2009-05-12;69(10):4537-44.
Species: Mouse
Sample Types: In Vivo
Applications: In Vivo
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