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

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.

• Reactivity: Pm-Cm
• Applications: Enzyme Activity
• Format: Carrier-Free

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

• Details of Functionality: 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 #: XP_005580845.2
• N-terminal Sequence: No results obtained. Gln21 is inferred from enzymatic pyroglutamate treatment revealing Asp22.
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 21 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: 21-27 kDa, reducing conditions

Packaging, Storage & Formulations

• 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.
• Buffer: Supplied as a 0.2 μm filtered solution in MES and NaCl.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • 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

Notes

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

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

• 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
• Uterocalin

Background

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).

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.

Publications for Lipocalin-2/NGAL (2357-LC)(1)

Publication using 2357-LC

• A Bashir, Q Yang, J Wang, S Hoyer, W Chou, C McLean, G Davis, Q Gong, Z Armstrong, J Jang, H Kang, A Pawlosky, A Scott, GE Dahl, M Berndl, M Dimon, BS Ferguson Machine learning guided aptamer refinement and discovery Nature Communications, 2021-04-22;12(1):2366. 2021-04-22 [PMID: 33888692] (Flow Cytometry, N/A)

Bioinformatics

• Uniprot:
  • XP_005580845.2()