Recombinant Mouse Fetuin A/AHSG Protein, CF Summary
Product Specifications
Ala19-Ile345, 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.
1563-PI
| 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 Procedure
- Assay Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse Fetuin A/AHSG (rmFetuin A) (Catalog # 1563-PI)
- Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhTrypsin 3 to 0.6 µg/mL in Assay Buffer.
- Prepare a curve of rmFetuin A (MW: 36651 Da) in Assay Buffer. Make the following serial dilutions: 6500, 1952, 651, 195.4, 65.1, 19.6, and 6.5 nM.
- Combine 20 µL of 0.6 µg/mL rhTrypsin 3, 100 µL of each dilution from the rmFetuin A curve, and 80 µL of Assay Buffer. Include a control (in duplicate) containing 180 µL of Assay Buffer and 20 µL of the diluted rhTrypsin 3.
- Incubate mixtures at room temperature for 30 minutes.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the incubated mixtures into a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibition concentration (IC50) value for rmFetuin A by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
- The specific activity for rhTrypsin 3 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rhTrypsin 3: 0.003 µg (1.17 nM)
- rmFetuin A curve: 1625, 488, 163, 48.9, 16.3, 4.9, and 1.625 nM
- Substrate: 10 µM
Reconstitution Calculator
Background: Fetuin A/AHSG
Mouse Fetuin A, also known as alpha 2-Heremans-Schmid glycoprotein, is encoded by the AHSG gene. It has been also called "countertrypsin" because of its ability to inhibit trypsin (1). It is a major plasma protein and a member of the cystatin superfamily of protease inhibitors (2, 3). It is expressed by hepatocytes, the principal cell source, and by monocyte/macrophages (4). The major form of plasma Fetuin A corresponds to two disulfide bond-linked chains derived from the single chain (5). 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 (7, 8).
- Yamamoto, K. and H. Sinohara (1993) J. Biol. Chem. 268:17750.
- 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 Mouse 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.
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Citations: Showing 1 - 4
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Fetuin-A alleviates neuroinflammation against traumatic brain injury-induced microglial necroptosis by regulating Nrf-2/HO-1 pathway
Authors: P Zhao, Y Wei, G Sun, L Xu, T Wang, Y Tian, H Chao, Y Tu, J Ji
Oncogene, 2022-11-04;19(1):269.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Adipocyte fetuin-A contributes to macrophage migration into adipose tissue and polarization of macrophages.
Authors: Chatterjee, Priyajit, Seal, Soma, Mukherjee, Sandip, Kundu, Rakesh, Mukherjee, Sutapa, Ray, Sukanta, Mukhopadhyay, Satinath, Majumdar, Subeer S, Bhattacharya, Samir
J Biol Chem, 2013-08-13;288(39):28324-30.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance.
Nat Med, 2012-07-29;18(0):1279.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay -
NF-kappaB mediates lipid-induced fetuin-A expression in hepatocytes that impairs adipocyte function effecting insulin resistance.
Authors: Dasgupta S, Bhattacharya S, Biswas A
Biochem. J., 2010-08-01;429(3):451-62.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay
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