• 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 calcium phosphate precipitation. Schinke, T.     et al. (1996) J. Biol. Chem.     271:20789. The IC    ₅₀ value is<50 μg/mL, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com  

• Source

  Mouse myeloma cell line, NS0-derived Ala19-Val367, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala19

• Predicted Molecular Mass

  39 kDa

• SDS-PAGE

  58 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Hepes, pH 7.5

• Recombinant Human Fetuin A/AHSG (rhFetuin A) (Catalog # 1184-PI)

• Calcium chloride (Sigma, Catalog # C3881), 100 mM stock in deionized water

• Sodium phosphate, pH 7.5 (Sigma, Catalog # S9638), 100 mM stock in deionized water

• Acetic acid (Sigma, Catalog # 242853), 100 mM stock in deionized water

• Malachite Green Phosphate Detection Kit (Catalog # )

• 96 well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute sodium phosphate to 3.2 mM in Assay buffer.

2. Dilute calcium chloride to 9.6 mM in Assay Buffer.

3. Dilute rhFetuin A to 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95, and 0.97 μg/mL in Assay Buffer containing 3.2 mM sodium phosphate.

4. Combine 50 μL of dilute calcium chloride with 50 μL of each dilution of rhFetuin A. Include an enzyme blank containing 50 μL calcium chloride and 50 μL dilute sodium phosphate buffer.

5. Incubate reactions at 37 °C for 100 minutes.

6. Centrifuge reactions in a tabletop microcentrifuge at maximum speed for 5 minutes.

7. Carefully remove the supernatant from each tube, and discard.

8. Resuspend each pellet in 200 μL of 100 mM acetic acid. Vortex briefly.

9. Incubate suspension at room temperature for 15 minutes.

10. Add 800 μL of deionized water to each tube.

11. Load 50 μL of each reaction into wells of a 96 well plate.

12. Add 25 μL of the Malachite Green Reagent A to all wells.  Mix briefly.

13. Add 75 μL of deionized water to all wells. Mix briefly.

14. Add 25 μL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.

15. Read plate at 620 nm (absorbance) in endpoint mode.

16. Derive the 50% inhibiting concentration (IC₅₀) of rhFetuin A by plotting absorbance vs. reaction concentration of rhFetuin A with 4?PL fitting.

Per Incubation:

• rhFetuin A curve: 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.95, 0.97 and 0.49 μg/mL

• Calcium chloride: 4.8 mM

• Sodium phosphate: 1.6 mM

Background: Fetuin A/AHSG

Human Fetuin A, also known as alpha   ₂-Heremans-Schmid glycoprotein, is encoded by the AHSG gene. It is a major plasma protein and a member of the cystatin superfamily of protease inhibitors (1, 2). It is expressed by hepatocytes, the principal cell source, and by monocyte/macrophages (3). The major form of plasma Fetuin A corresponds to a disulfide bond-linked two chains derived from the single chain (4). Human Fetuin A has a number of functions. It is a negative acute-phase protein with normal circulating levels in adults (300?600 μg/mL), which fall significantly (30?50%) during injury and infection (5). It enhances entry of cationic inhibitors into macrophages (6). It inhibits both insulin receptor autophosphorylation and undesirable calcification (6, 7). The purified recombinant human Fetuin A corresponds to the single chain, which can be converted to the two-chain form by recombinant human Furin (Catalog # )   in vitro. However, the conversion does not enhance its inhibitory activity against recombinant human Cathpesin V, a cysteine protease.

• References:

1. Kellemann, J. et al. (1989) J. Biol. Chem. 264:14121.

2. Dziegielewska, K.M. et al. (1990) J. Biol. Chem. 265:4354.

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

4. Gejyo, F. and K. Schmid (1981) Biochim. Biophys. Acta. 671:78.

5. Wang, H. et al. (1998) Proc. Natl. Acad. Sci. USA 95:14429.

6. Mathews, S.T. et al. (2000) Mol. Cell Endocrinol. 164:87.

7. Sch?fer, C. et al. (2003) J. Clin. Invest. 112:357.

• Long Name:

  alpha-2-HS Glycoprotein

• Entrez Gene IDs:

  197 (Human); 11625 (Mouse); 25373 (Rat)

• Alternate Names:

  A2HS; AHS; AHSG; alpha-2-HS-glycoprotein; alpha-2-Z-globulin; Ba-alpha-2-glycoprotein; FETUAba-alpha-2-glycoprotein; Fetuin A; fetuin-A; HSGA