• 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 active Cathepsin V cleavage of a fluorogenic peptide substrate Z-LR-AMC (Catalog # ). The IC    ₅₀ value is<150 nM, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com  

• Source

  Mouse myeloma cell line, NS0-derived Met19-Pro382, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met19

• Predicted Molecular Mass

  42 kDa

• SDS-PAGE

  54 kDa and 58 kDa, reducing conditions

Assay Procedure

Materials

• 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 # )

• Substrate: Z-Leu-Arg-AMC (Catalog # )

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCathepsin V to 2 µg/mL in Assay Buffer.

2. 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.

3. 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.

4. Incubate mixtures at room temperature for 30 minutes.

5. Dilute Substrate to 20 µM in Assay Buffer.

6. Load into a black well plate 50 µL of the incubated mixtures and start the reaction by adding 50 µL of 20 µM Substrate.

7. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.

8. Derive the 50% inhibition concentration (IC₅₀) for rhFetuin B by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.

9. The specific activity for rhCathepsin V at each point may be determined using the following formula (if needed):

     *Adjusted for Substrate Blank
     **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

Per Well:

• 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).

• References:

1. Oliver, E., et al. (1999) Genomics. 57:352.

2. Oliver, E., et al. (2000) Biochem. J. 350:589.

3. Kellemann, J. et al., 1989, J. Biol. Chem. 264:14121.

4. Denecke, B. et al. (2003) Biochem. J. 376:135.

5. Schäfer, C., et al. (2003) J. Clin. Invest. 112:357.

6. Dziegielewska, K. M., et al. (1996) Histochem. Cell Biol. 106:319.

7. Gangneux, C. et al. (2003) Nucleic Acids Res. 31:5957.

• Entrez Gene IDs:

  26998 (Human)

• Alternate Names:

  FETUB; Fetuin B