Recombinant Cynomolgus Lipocalin-2/NGAL His-tag Protein, CF Summary
with C-terminal 6-His tag
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.
|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 deionized water.
- 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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