Recombinant Cynomolgus Lipocalin-2/NGAL His-tag Protein, CF

Catalog # Availability Size / Price Qty
2357-LC-050
Lipocalin-2/NGAL Protein Enzyme Activity
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Recombinant Cynomolgus Lipocalin-2/NGAL His-tag Protein, CF Summary

Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<0.10 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to bind Iron(III) dihydroxybenzoic acid [Fe(DHBA)3]. The binding of Fe(DHBA)3 results in the quenching of Trp fluorescence in Lipocalin-2. >1.0 μM of Fe(DHBA)3 can be bound under the described conditions.
Source
Chinese Hamster Ovary cell line, CHO-derived cynomolgus monkey Lipocalin-2/NGAL protein
Gln21-Gly198
with C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
No results obtained. Gln21 is inferred from enzymatic pyroglutamate treatment revealing Asp22.
Predicted Molecular Mass
21 kDa
SDS-PAGE
21-27 kDa, reducing conditions

Product Datasheets

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

2357-LC

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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials
  •  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
  1. 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.
  2. Prepare 1 mM DHBA in Ligand Buffer from powder stock.
  3. Combine equal volumes of the Fe3+ curve with 1 mM DHBA. Include a control containing 1 mM DHBA and deionized water.
  4. Incubate at room temperature for 10 minutes. A curve of the metal ligand complex of Fe(DHBA)3 is formed.
  5. After incubation, perform 5 fold dilutions to the curve using Assay Buffer.
  6. Dilute rcynoLipocalin-2 (MW: 21200 Da) to 4 µM in Assay Buffer.
  7. In the plate, load 50 µL of the diluted Fe(DHBA)3 complex curve and 50 µL of 4 µM rcynoLipocalin-2.
  8. Incubate at room temperature for 30 minutes.
  9. Read at excitation and emission wavelengths of 280 nm and 340 nm, respectively in endpoint mode.
  10. Plot a 4-parameter curve of Fe(DHBA)3 Concentration (x-axis) versus RFUs ( gamma -axis), and calculate a BC50 from the curve.
Per Well:
  • 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

Data Images

Binding Activity Binding Activity View Larger

Recombinant Cynomolgus Monkey Lipocalin-2 (Catalog # 2357-LC) is measured by itsability to bind Iron(III) dihydroxybenzoic acid.

SDS-PAGE SDS-PAGE View Larger

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.

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Background: Lipocalin-2/NGAL

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).

References
  1. Flower, D. R. et al. (1994) FEBS Lett. 354:7.
  2. Kjeldsen L. et al. (2000) Biochim. Biophys. Acta. 1482:272.
  3. Goetz, D. H. et al. (2002) Mol. Cell 10:1033.
  4. Flo, T. H. et al. (2004) Nature 432:917.
  5. Yang, M. B. et al. (2002) Mol. Cell. 10:1045.
  6. Ferreira, A. C. et al. (2018) Neurosci. BioBehav. Rev. 95:73.
  7. Devireddy, L. R. et al. (2001) Science 293:829.
  8. Jung, M. et al. (2017) Front. Immunol. 8:1171.
  9. Hanai, J. et al. (2005) J. Biol. Chem. 280:13641.
  10. Mori, K. et al. (2005) J. Clin. Invest. 115:610.
  11. Sun, W. Y. et al. (2018) JCI Insight. 3:120196.
  12. Zhou, F. et al. (2016) Eur. J. Cardiothorac. Surg. 49:746.
Long Name
Neutrophil Gelatinase-associated Lipocalin
Entrez Gene IDs
3934 (Human); 16819 (Mouse); 170496 (Rat); 102137149 (Cynomolgus Monkey)
Alternate Names
24p3; 25 kDa alpha-2-microglobulin-related subunit of MMP-9; HNL; LCN2; lipocalin 2 (oncogene 24p3); lipocalin 2; Lipocalin2; Lipocalin-2; migration-stimulating factor inhibitor; MSFI; neutrophil gelatinase-associated lipocalin; NGAL; NGALlipocalin-2; Oncogene 24p3; p25; Siderocalin

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