Recombinant Human Fetuin A/AHSG Protein, CF Summary
Ala19-Val367, 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||Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.|
|Reconstitution||Reconstitute at 500 μg/mL in sterile 25 mM Tris and 150 mM NaCl, pH 7.5.|
|Shipping||The product is shipped at ambient temperature. 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: 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 # DY996)
- 96 well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute sodium phosphate to 3.2 mM in Assay buffer.
- Dilute calcium chloride to 9.6 mM in Assay Buffer.
- 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.
- 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.
- Incubate reactions at 37 °C for 100 minutes.
- Centrifuge reactions in a tabletop microcentrifuge at maximum speed for 5 minutes.
- Carefully remove the supernatant from each tube, and discard.
- Resuspend each pellet in 200 µL of 100 mM acetic acid. Vortex briefly.
- Incubate suspension at room temperature for 15 minutes.
- Add 800 µL of deionized water to each tube.
- Load 50 µL of each reaction into wells of a 96 well plate.
- Add 25 µL of the Malachite Green Reagent A to all wells. Mix briefly.
- Add 75 µL of deionized water to all wells. Mix briefly.
- Add 25 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- Derive the 50% inhibiting concentration (IC50) of rhFetuin A by plotting absorbance vs. reaction concentration of rhFetuin A with 4‑PL fitting.
- 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 # 1503-SE) in vitro. However, the conversion does not enhance its inhibitory activity against recombinant human Cathpesin V, a cysteine protease.
- Kellemann, J. et al. (1989) J. Biol. Chem. 264:14121.
- Dziegielewska, K.M. et al. (1990) J. Biol. Chem. 265:4354.
- Dziegielewska, K.M. et al. (1996) Histochem. Cell Biol. 106:319.
- Gejyo, F. and K. Schmid (1981) Biochim. Biophys. Acta. 671:78.
- Wang, H. et al. (1998) Proc. Natl. Acad. Sci. USA 95:14429.
- Mathews, S.T. et al. (2000) Mol. Cell Endocrinol. 164:87.
- Schäfer, C. et al. (2003) J. Clin. Invest. 112:357.
Citations for Recombinant Human Fetuin A/AHSG 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 - 2
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Association of fetuin-A with incident type 2 diabetes: Results from the MONICA/KORA Augsburg study and a systematic meta-analysis
Authors: C Sujana, C Huth, A Zierer, S Meesters, J Sudduth-Kl, W Koenig, C Herder, A Peters, B Thorand
Eur. J. Endocrinol., 2018;0(0):.
Sample Types: Serum
Applications: Single Molecule Capture Standa
Nanobacteria are mineralo fetuin complexes.
Authors: Raoult D, Drancourt M, Azza S, Nappez C, Guieu R, Rolain JM, Fourquet P, Campagna B, La Scola B, Mege JL, Mansuelle P, Lechevalier E, Berland Y, Gorvel JP, Renesto P
PLoS Pathog., 2008;4(2):e41.
Sample Types: N/A
Applications: ELISA (Standard)
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