Recombinant Mouse Fetuin A/AHSG Protein, CF

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Product Details

Summary
Reactivity MuSpecies Glossary
Applications Inhibition Activity
Format
Carrier-Free

Order Details

Recombinant Mouse Fetuin A/AHSG Protein, CF Summary

Details of Functionality
Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002). The IC50 value is <0.20 µM, as measured under the the described conditions.
Source
Mouse myeloma cell line, NS0-derived mouse Fetuin A/AHSG protein
Ala19-Ile345, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
Ala19
Protein/Peptide Type
Recombinant Enzymes
Gene
Ahsg
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Theoretical MW
37 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
58 kDa, reducing conditions
Publications
Read Publications using
1563-PI in the following applications:

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
Buffer
Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Reconstitution Instructions
Reconstitute at 500 μg/mL in sterile 25 mM Tris and 150 mM NaCl, pH 7.5.
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
  1. Dilute rhTrypsin 3 to 0.6 µg/mL in Assay Buffer.
  2. 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.
  3. 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.
  4. Incubate mixtures at room temperature for 30 minutes.
  5. Dilute Substrate to 20 µM in Assay Buffer.
  6. Load 50 µL of the incubated mixtures into a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
  7. Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
  8. Derive the 50% inhibition concentration (IC50) value for rmFetuin A by plotting RFU/min (or specific activity) vs. concentration with 4‑PL fitting.
  9. 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).

Per Well:
  • 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Fetuin A/AHSG Protein, CF

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

Background

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).

  1. Yamamoto, K. and H. Sinohara (1993) J. Biol. Chem. 268:17750.
  2. Kellemann, J. et al. (1989) J. Biol. Chem. 264:14121.
  3. Dziegielewska, K.M. et al. (1990) J. Biol. Chem. 265:4354.
  4. Dziegielewska, K.M. et al. (1996) Histochem. Cell Biol. 106:319.
  5. Gejyo, F. and K. Schmid (1981) Biochim. Biophys. Acta 671:78.
  6. Wang, H. et al. (1998) Proc. Natl. Acad. Sci. USA 95:14429.
  7. Mathews, S.T. et al. (2000) Mol. Cell Endocrinol. 164:87.
  8. Schäfer, C. et al. (2003) J. Clin. Invest. 112:357.

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1563-PI
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Applications: Inhibition Activity

Publications for Fetuin A/AHSG (1563-PI)(3)

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Bioinformatics

Gene Symbol Ahsg
Uniprot