• 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. Recombinant Rat Lipocalin?2/NGAL can bind >1.0 μM of Fe(DHBA)    ₃, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Mouse myeloma cell line, NS0-derived Gln21-Asn198, with a C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  No results obtained: Gln21 predicted

• Predicted Molecular Mass

  21 kDa

• SDS-PAGE

  27 kDa, reducing conditions

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 Rat Lipocalin-2/NGAL (rrLipocalin-2) (Catalog # 3508-LC)

• Iron (III) (Fe(III)) (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 Fe(III) in deionized water with the following serial dilutions: 640, 320, 160, 80, 40, 20, 10, 5, and 2.5 μM.

2. Weigh DHBA (MW: 154.12 g/mol) in grams and dissolve it with an appropriate volume of Ligand Buffer for a 10 mM concentration.

3. Further dilute DHBA to a concentration of 1 mM.

4. Mix equal volumes of the diluted Fe(III) curve and diluted DHBA. Include a control containing deionized water and diluted DHBA only.

5. Incubate at room temperature for 10 minutes.

6. Perform a 1/5 dilution to the incubated Fe(III)(DHBA)₃ reaction mixtures with Assay Buffer.

7. Dilute rrLipocalin-2 (21,304 Da) to 4 μM with Assay Buffer.

8. Load 50 μL of the diluted Fe(DHBA)₃ curve with 50 μL of 4 μM rrLipocalin-2.

9. Incubate plate at room temperature for 30 minutes.

10. Read at excitation and emission wavelengths of 280 nm and 340 nm (top read), respectively, in endpoint mode.

11. Create a plot of Fe(DHBA)₃ (x-axis) versus RFUs (Raw Data) (y-axis), and determine a BC₅₀ from the curve using 4-PL fitting.

Per Well:

• rrLipocalin-2/NGAL: 2 μM

• Fe(DHBA)₃ Curve: 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 μM

Background: Lipocalin-2/NGAL

Lipocalin-2, also known as neutrophil gelatinase-associated Lipocalin and uterocalin (NGAL), has been implicated in a variety of processes including cell differentiation, tumorigenesis, and apoptosis (1?3). It binds a bacterial catecholate sidropore bound to ferric ion such as enterobactin with a subnanomolar dissociation constant (K_D = 0.41 nM) (4). 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) (4). Secretion of Lipocalin?2 in immune cells increases by stimulation of Toll?like receptor as a acute phase response to infection. As a result, it acts as a potent bacteriostatic reagents by sequestering iron (5). 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 (6). In the kidney, Lipocalin?2?mediated iron trafficking may be involved in protection from renal injury, and it has been implicated as a marker for early kidney failure (7, 8).

• References:

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

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

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

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

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

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

7. Mori, K. et al. (2005) J. Clin. Invest. 115:610.

8. Mishra, J. et al. (2005) Lancet 365:1231.

• 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