Recombinant Human IL-4I1 Protein, CF Summary
Product Specifications
Gln22-His567, with a C-terminal 6-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.
5684-AO
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with polar packs. 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 Sodium Phosphate, pH 7.0
- Recombinant Human IL-4I1 (rhIL-4I1) (Catalog # 5684-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
- Dilute rhIL-4I1 to 2 ng/µL in Assay Buffer.
- Prepare the Substrate mixture including 2 mM Phenylalanine, 2 units/mL HRP and 100 µM AUR in Assay Buffer.
- Load in a black well plate 50 µL of 2 ng/µL of rhIL-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.
- 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.
- 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.
- rhIL-4I1: 0.1 µg
- Phenylalanine: 1 mM
- HRP: 1 unit/mL
- AUR: 50 µM
Caution: The rhIL-4I1 enzymatic reaction may significantly be interfered with organic buffer such as MES.
Scientific Data
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Recombinant Human IL-4I1 Protein (Catalog # 5684-AO) is measured by its ability to oxidize phenylalanine in a horseradish peroxidase coupled assay.
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2 μg/lane of Recombinant Human IL-4I1 Protein (5684-AO) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at ~71 under reducing conditions.
Reconstitution Calculator
Background: IL-4I1
Interleukin 4 induced protein 1 (IL-4I1), also known as protein FIG-1 and L-amino acid oxidase, is encoded by a B-cell IL-4-inducible gene, FIG1, and is highly expressed in primary metastinal B-cell lymphomas (1-4). It belongs to the flavin monoamine oxidase family, FIG1 subfamily. Enzymological characterization reveals that IL-4I1 has L-amino acid oxidase activity with preference toward aromatic amino acids. Studies have shown that hIL-4I1 inhibited the proliferation of CD3‑stimulated T lymphocytes with a similar effect on CD4(+) and CD8(+) T cells (5). Its inhibitory effect was dependent on enzymatic activity and H2O2 production. Its restricted expression to lymphoid tissues indicates that it may play an important function in the immune system (1, 4).
- Chu, C.C. and W.E. Paul. (1997) Proc. Natl. Acad. Sci. USA 94:2507.
- Mason, J.M. et al. (2004) J. Immunol. 173:4561.
- Chavan, S.S. et al. (2002) Biochim. Biophys. Acta. 1576:70.
- Copie-Bergman, C. et al. (2003) Blood 101:2756.
- Boulland, M.L. et al. (2007) Blood 110:220.
Citation for Recombinant Human IL-4I1 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.
1 Citation: Showing 1 - 1
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Identification of inhibitors of the immunosuppressive enzyme IL4I1
Authors: M Presset, D Djordjevic, A Dupont, E Le Gall, V Molinier-F, F Castellano
Bioorg. Chem., 2019-11-23;94(0):103463.
Species: Human
Sample Types: Protein
Applications: Bioassay
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