• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 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. rhLipocalin-2 can bind >1.5 μM of Fe(DHBA)    ₃ under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Gln21-Gly198, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  No results obtained: Gln21 predicted

• Predicted Molecular Mass

  22 kDa

• SDS-PAGE

  25-27 kDa doublet, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)

• Recombinant Human Lipocalin?2/NGAL (rhLipocalin-2) (Catalog # 1757-LC)

• Iron III (Fe³⁺) (Sigma, Catalog # 16596)

• 2,3,Dihydroxybenzoic Acid (DHBA) (Sigma, Catalog # 126209)

• Ligand Buffer: 0.1 M Tris, pH 8.0

• 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 Ligand Buffer.

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 rhLipocalin-2 (MW: 21905 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 rhLipocalin-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 (y-axis), and calculate a BC₅₀ from the curve.

Per Well:

• Fe(DHBA)₃ Curve: 0.001, 0.125, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, 16, and 32 μM.

• rhLipocalin-2: 2 μM

Background: Lipocalin-2/NGAL

Members of Lipocalin family share a highly conserved fold with an eight-stranded antiparallel beta barrel, and act as a transporters, carrying small molecules to specific cells (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 human neutrophil gelatinases (MMP-9) (2). Its expression has been observed in most tissues normally exposed to microorganism, and its synthesis is induced in epithelial cells during inflammation (3). Lipocalin-2 has been implicated in a variety of processes including cell differentiation, tumorigenesis, and apoptosis (3?5). Studies indicate that Lipocalin-2 binds a bacterial catecholate sidropore bound to ferric ion such as enterobactin with a subnanomolar dissociation constant (K_d = 0.41 nM) (6). The bound ferric enterobactin complex breaks down slowly in a month into dihydroxybenzoyl serine and dihydroxybenzoic acid (DHBA). It also binds to a ferric DHBA complex with much less K_d values (7.9 nM) (6). 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 (7). Moreover, Lipocalin-2 can alter the invasive and metastatic behavior of Ras-transformed breast cancer cells in vitro and in vivo by reversing epithelial to mesenchymal transition inducing activity of Ras, through restoration of E-cadherin expression, via effects on the Ras-MAPK signaling pathway (8).

• References:

1. Flower, D.R. et al. (1994) FEBS Lett. 354:7.

2. Kjeldsen, L. et al. (1993) J. Biol. Chem. 268:10426.

3. Kjeldsen L, et al. (2000) Biochim Biophys Acta. 1482:272.

4. Devireddy, L.R. et al. (2001) Science 293: 829.

5. Yang, M.B. et al. (2002) Mol. Cell. 10:1045.

6. Goetz, D.H. et al. (2002) Mol. Cell 10:1033.

7. Flo, T.H. et al. (2004) Nature 432:917.

8. Hanai, J. et al. (2005) J. Biol. Chem. 280:13641.

• Long Name:

  Neutrophil Gelatinase-associated Lipocalin

• Entrez Gene IDs:

  3934 (Human); 16819 (Mouse); 170496 (Rat)

• 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