Recombinant Human Fetuin B Protein, CF Summary
Met19-Pro382, with a C-terminal 10-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||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|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 Buffer: 25 mM Sodium Acetate, 0.10 M NaCl, 5 mM DTT, pH 5.5
- Recombinant Human Fetuin B (rhFetuin B) (Catalog # 1725-PI)
- Recombinant Human Cathepsin V (rhCathepsin V) (Catalog # 1080-CY)
- Substrate: Z-Leu-Arg-AMC (Catalog # ES008)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhCathepsin V to 2 µg/mL in Assay Buffer.
- Prepare a curve of rhFetuin B (MW: 41601 Da) in Assay Buffer. Make the following serial dilutions: 6000, 3000, 1500, 500, 250, 150, 100, 50, 25 and 2.5 nM.
- Mix equal volumes of the rhFetuin B curve dilutions and the diluted rhCathepsin V. Include a control (in duplicate) containing Assay Buffer and the diluted active rhCathepsin V.
- Incubate mixtures at room temperature for 30 minutes.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load into a black well plate 50 µL of the incubated mixtures and start the reaction by adding 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibition concentration (IC50) for rhFetuin B by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for rhCathepsin V 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 (Sigma, Catalog # A-9891).
- rhCathepsin V: 0.050 µg
- rhFetuin B curve: 1500, 750, 375, 125, 62.5, 37.5, 25, 12.5, 6.25 and 0.625 nM
- Substrate: 10 µM
Background: Fetuin B
Fetuins are members of the cystatin superfamily of cysteine protease inhibitors (1-3). Additional members of this superfamily are kininogen and histidine-rich glycoprotein. Fetuin A and B are two known members of the fetuin family. Hepatocytes are believed to be the principal cellular source, but other cell types also express it (4, 5). Fetuin A, also known as alpha 2-Heremans-Schmid glycoprotein, is an inhibitor of basic calcium phosphate precipitation and a negative acute-phase protein (6, 7). Normal circulating levels of Fetuin A in adults (300-600 μg/mL) fall significantly (30-50%) during injury and infection (7). Fetuin B is a newer member whose function is not fully characterized (1, 2). Fetuin A and B display similarities and differences in their characteristics. Fetuin B exhibits reduction of calcification, while both mRNA levels were down‑regulated during the acute phase in inflammation-induced rats (4). However, they share only 20% amino acid sequence identity (2). The amounts of Fetuin B in human serum, unlike Fetuin A, vary with gender and are higher in females than in males (4).
- Oliver, E., et al. (1999) Genomics. 57:352.
- Oliver, E., et al. (2000) Biochem. J. 350:589.
- Kellemann, J. et al., 1989, J. Biol. Chem. 264:14121.
- Denecke, B. et al. (2003) Biochem. J. 376:135.
- Schäfer, C., et al. (2003) J. Clin. Invest. 112:357.
- Dziegielewska, K. M., et al. (1996) Histochem. Cell Biol. 106:319.
- Gangneux, C. et al. (2003) Nucleic Acids Res. 31:5957.
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Fluorogenic Peptide Substrates
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