Recombinant Mouse IL-4I1 Protein, CF

• Reactivity: Mu
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant Mouse IL-4I1 Protein, CF Summary

• Details of Functionality: Measured by its ability to produce hydrogen peroxide during the oxidation of phenylalanine. The specific activity is >100 pmol/min/μg, as measured under the described conditions.
• Source: Chinese Hamster Ovary cell line, CHO-derived mouse IL-4I1 protein
  Leu21-Ser630, with a C-terminal 6-His tag
• Accession #: NP_034345
• N-terminal Sequence: Leu21
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Il4i1
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
• Theoretical MW: 69 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: 86 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 0.1 M Sodium Phosphate, pH 8.0
  • Recombinant Mouse IL-4I1 (rmIL-4I1) (Catalog # 5576-AO)
  • Coupling Agent: Horseradish Peroxidase (HRP) (250-330 U/mg) (Sigma, Catalog # P8375), 250 units/mL stock in 0.1 M Sodium Phosphate, pH 8.0
  • Substrate Component 1: Phenylalanine (Sigma, Catalog # P2126), 100 mM stock in diH₂O
  • Substrate Component 2: Amplex Ultra Red (AUR) (Molecular Probes, Catalog # A36006), 10 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 rmIL-4I1 to 2 ng/µL in Assay Buffer.
  2. Prepare the Substrate mixture including 2 mM Phenylalanine, 2 units/mL HRP and 100 µM AUR in Assay Buffer.
  3. Load in a black well plate 50 µL of 2 ng/µL of rmIL-4I1, and start the reaction by adding 50 µL of the Substrate mixture (step 2). Include a Substrate Blank containing 50 µL of the Assay Buffer and 50 µL of the Substrate mixture.
  4. Read at excitation and emission wavelengths of 544 nm and 590 nm (top read), respectively in kinetic mode for 5 minutes. Note: A wavelength cutoff of 570 nm should be manually specified.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using a fluorescent standard prepared by incubating 50 µM AUR, 1 unit/mL HRP, 1 mM Phenylalanine, and a range of Hydrogen Peroxide (0-500 pmol) (Sigma, Catalog # H1009) in Assay Buffer at room temperature. Use the resulting amounts of oxidized AUR to determine the conversion factor.

  Per Well:
  • rmIL-4I1: 0.1 µg
  • Phenylalanine: 1 mM
  • HRP: 1 unit/mL
  • AUR: 50 µM

  Caution: The rmIL-4I1 enzymatic reaction may significantly be interfered with organic buffer such as MES.

Notes

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

Alternate Names for Recombinant Mouse IL-4I1 Protein, CF

• Fig-1 protein
• FIG1
• hFIG1
• IL4I1
• IL-4I1
• IL4-induced protein 1
• interleukin 4 induced 1
• interleukin four induced 1
• LAAO
• L-amino-acid oxidase
• LAO
• UNQ636/PRO1265

Background

Interleukin four-induced gene 1 (IL-4I1) was first identified in mouse B cells as part of the response to IL-4, a key regulator of the immune response (1). Later, the enzyme was shown to be a member of the flavin-containing amino acid oxidase family and closely related to L-amino acid oxidases (2, 3). Enzymological characterization reveals that IL-4I1 has L-amino acid oxidase activity with preference toward aromatic amino acids (4). Its limited expression in immune tissues, dendritic cells, and macrophages indicates that it may play an important function in the immune system (5, 6).

1. Chu, C.C. and W.E. Paul (1997) Proc. Natl. Acad. Sci. USA 94:2507.
2. Raibekas, A.A. and V. Massey (1998) Biochem. Biophys. Res. Comm. 248:476.
3. Chavan, S.S. et al. (2002) Biochim. Biophys. Acta 1576:70.
4. Mason, J.M. et al. (2004) J. Immunol. 173:4561.
5. Copie-Berman, C. et al. (2003) Blood 101:2756.
6. Bolland, M.-L. et al. (2007) Blood 110:220.

Publications for IL-4I1 (5576-AO)(2)

Publications using 5576-AO

• L Bod, L Douguet, C Auffray, R Lengagne, F Bekkat, E Rondeau, V Molinier-F, F Castellano, Y Richard, A Prévost-Bl IL-4-Induced Gene 1: A Negative Immune Checkpoint Controlling B Cell Differentiation and Activation J. Immunol., 2017-12-29;0(0):. 2017-12-29 [PMID: 29288206] (Bioassay, Mouse)
• L Bod, R Lengagne, L Wrobel, JP Ramspott, M Kato, MF Avril, F Castellano, V Molinier-F, A Prévost-Bl IL4-induced gene 1 promotes tumor growth by shaping the immune microenvironment in melanoma Oncoimmunology, 2017-01-13;6(3):e1278331. 2017-01-13 [PMID: 28405502] (Bioassay, Mouse)

Bioinformatics

• Gene Symbol: Il4i1
• Uniprot:
  • NP_034345()