Lipocalin-2, also known as Neutrophil Gelatinase-Associated Lipocalin (NGAL), was originally identified as a component of neutrophil granules. 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). Its expression has been observed in most tissues normally exposed to microorganism, and its synthesis is induced in epithelial cells during inflammation. Lipocalin-2 has been implicated in a variety of processes including cell differentiation, tumorigenesis, and apoptosis.?Studies indicate that Lipocalin-2 binds a bacterial catecholate sidropore bound to ferric ion such as enterobactin with a subnanomolar dissociation constant (Kd?= 0.41 nM). 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 Kd?values (7.9 nM). The activity assay of recombinant human NGAL was 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. The recombinant human NGAL was diluted to 100 ug/ml in assay buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5). 50 μL of the different concentrations of Fe(DHBA)3 and 50 μL of 100 ug/ml rmNGAL was loaded into the plate. Incubated at room temperature for 30 minutes, then read at excitation and emission wavelengths of 280 nM and 340 nM in endpoint mode. The result was shown in Figure 1, the recombinant human Lipocalin-2 can bind >8 μM of Fe(DHBA)3.