Recombinant Cynomolgus Lipocalin-2/NGAL His-tag Protein, CF Summary
with C-terminal 6-His tag
|Formulation||Supplied as a 0.2 μm filtered solution in MES and NaCl.|
|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 Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
- Ligand Buffer: 0.1 M Tris, pH 8.0
- Recombinant Cynomolgus Monkey Lipocalin‑2/NGAL His-tag (cynoLipocalin-2) (Catalog # 2357-LC)
- Iron III (Fe3+) (Sigma, Catalog # 16596)
- 2,3,Dihydroxybenzoic Acid (DHBA) (Sigma, Catalog # 126209)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Prepare a curve of Fe3+ in deionized water with the following serial dilutions: 640, 320, 160, 80, 40, 20, 10, 5, and 2.5 µM.
- Prepare 1 mM DHBA in Ligand Buffer from powder stock.
- Combine equal volumes of the Fe3+ curve with 1 mM DHBA. Include a control containing 1 mM DHBA and Ligand Buffer.
- Incubate at room temperature for 10 minutes. A curve of the metal ligand complex of Fe(DHBA)3 is formed.
- After incubation, perform 5 fold dilutions to the curve using Assay Buffer.
- Dilute rcynoLipocalin-2 (MW: 21200 Da) to 4 µM in Assay Buffer.
- In the plate, load 50 µL of the diluted Fe(DHBA)3 complex curve and 50 µL of 4 µM rcynoLipocalin-2.
- Incubate at room temperature for 30 minutes.
- Read at excitation and emission wavelengths of 280 nm and 340 nm, respectively in endpoint mode.
- Plot a 4-parameter curve of Fe(DHBA)3 Concentration (x-axis) versus RFUs ( gamma -axis), and calculate a BC50 from the curve.
- Fe(DHBA)3 Complex Curve: 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16, and 32 µM.
- rcynoLipocalin-2: 2 µM
Recombinant Cynomolgus Monkey Lipocalin-2 (Catalog # 2357-LC) is measured by itsability to bind Iron(III) dihydroxybenzoic acid.
2 μg/lane of Recombinant Cynomolgus Monkey Lipocalin‑2/NGAL was resolved with SDS-PAGE underreducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Bluestaining, showing a primary band at 24 kDa under reducing conditions.
Lipocalin-2 (LCN-2) is a member of the lipocalin family. These proteins share a highly conserved fold with an eight-stranded antiparallel beta barrel and act as small molecule transporters (1). Lipocalin-2, also known as Neutrophil Gelatinase-Associated Lipocalin (NGAL), was originally identified as a component of neutrophil granules (2). It is a 25 kDa protein existing in monomeric and homo- and heterodimeric forms, the latter as a dimer with neutrophil gelatinases (MMP-9) (2). Studies indicate that Lipocalin-2 binds iron through association with dihydroxybenzoic acid (DHBA), a sidropore similar to bacterial enterobactin (3). Its expression has been observed in most tissues normally exposed to microorganisms and is induced in epithelial cells during inflammation (2). Secretion of Lipocalin-2 in immune cells increases by stimulation of Toll-like receptor as an acute phase response to infection. As a result, it acts as a potent bacteriostatic reagent by sequestering iron (4). Lipocalin-2 has been implicated in a variety of processes including cell differentiation, tumorigenesis, and apoptosis (5-8). Lipocalin-2 can alter the invasive and metastatic behavior of Ras-transformed breast cancer cells via effects on the Ras-MAPK signaling pathway (9). In the kidney, Lipocalin-2 mediated iron trafficking may be involved in renal injury, and it has been implicated as a marker for early kidney failure (10-12).
- Flower, D. R. et al. (1994) FEBS Lett. 354:7.
- Kjeldsen L. et al. (2000) Biochim. Biophys. Acta. 1482:272.
- Goetz, D. H. et al. (2002) Mol. Cell 10:1033.
- Flo, T. H. et al. (2004) Nature 432:917.
- Yang, M. B. et al. (2002) Mol. Cell. 10:1045.
- Ferreira, A. C. et al. (2018) Neurosci. BioBehav. Rev. 95:73.
- Devireddy, L. R. et al. (2001) Science 293:829.
- Jung, M. et al. (2017) Front. Immunol. 8:1171.
- Hanai, J. et al. (2005) J. Biol. Chem. 280:13641.
- Mori, K. et al. (2005) J. Clin. Invest. 115:610.
- Sun, W. Y. et al. (2018) JCI Insight. 3:120196.
- Zhou, F. et al. (2016) Eur. J. Cardiothorac. Surg. 49:746.
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