Recombinant Human Lipocalin-2/NGAL Protein, CF

• Reactivity: Hu
• Applications: Binding Activity
• Format: Carrier-Free

Recombinant Human Lipocalin-2/NGAL 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. rhLipocalin-2 can bind >1.5 µM of Fe(DHBA)₃ under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Lipocalin-2/NGAL protein
  Gln21-Gly198, with a C-terminal 10-His tag
• Accession #: P80188
• N-terminal Sequence: No results obtained: Gln21 predicted
• Protein/Peptide Type: Recombinant Enzymes
• Gene: LCN2
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Binding Activity
• Theoretical MW: 22 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: 25-27 kDa doublet, 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 under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • 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

Notes

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

Alternate Names for Recombinant Human Lipocalin-2/NGAL 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

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).

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.

Publications for Lipocalin-2/NGAL (1757-LC)(23)

Publications using 1757-LC

• W Santus, AP Rana, JR Devlin, KA Kiernan, CC Jacob, J Tjokrosurj, DM Underhill, J Behnsen Mycobiota and diet-derived fungal xenosiderophores promote Salmonella gastrointestinal colonization Nature Microbiology, 2022;7(12):2025-2038. 2022 [PMID: 36411353] (Bioassay, Salmonella)
• U Gupta, S Ghosh, CT Wallace, P Shang, Y Xin, AP Nair, M Yazdankhah, A Strizhakov, MA Ross, H Liu, S Hose, NA Stepicheva, O Chowdhury, M Nemani, V Maddipatla, R Grebe, M Das, KL Lathrop, JA Sahel, JS Zigler, J Qian, A Ghosh, Y Sergeev, JT Handa, CM St Croix, D Sinha Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD Autophagy, 2022;0(0):1-20. 2022 [PMID: 35473441] (Bioassay, Human)
• L Cook, M Sengelmann, B Winkler, C Nagl, S Koch, U Schlomann, EP Slater, MA Miller, EP von Strand, B Dörsam, C Preu beta er, JW Bartsch ADAM8-Dependent Extracellular Signaling in the Tumor Microenvironment Involves Regulated Release of Lipocalin 2 and MMP-9 International Journal of Molecular Sciences, 2022;23(4):. 2022 [PMID: 35216088] (Stimulation, Human)
• SK Schröder, M Pinoé-Schm, R Weiskirche Lipocalin-2 (LCN2) Deficiency Leads to Cellular Changes in Highly Metastatic Human Prostate Cancer Cell Line PC-3 Cells, 2022;11(2):. 2022 [PMID: 35053376] (Bioassay, Human)
• W Zhang, R Pan, M Lu, Q Zhang, Z Lin, Y Qin, Z Wang, S Gong, H Lin, S Chong, L Lu, W Liao, X Lu Epigenetic induction of lipocalin 2 expression drives acquired resistance to 5-fluorouracil in colorectal cancer through integrin beta3/SRC pathway Oncogene, 2021;0(0):. 2021 [PMID: 34588619] (Bioassay, Human)
• X Wen, B Su, M Gao, J Chen, D Zhou, H You, N Li, S Chang, X Cheng, C Qian, J Gao, P Yang, S Qu, L Bu Obesity-associated up-regulation of lipocalin 2 protects gastric mucosa cells from apoptotic cell death by reducing endoplasmic reticulum stress Cell Death & Disease, 2021;12(2):221. 2021 [PMID: 33637683] (Bioassay, Human)
• A Tyagi, S Sharma, K Wu, SY Wu, F Xing, Y Liu, D Zhao, RP Deshpande, RB D'Agostino, K Watabe Nicotine promotes breast cancer metastasis by stimulating N2 neutrophils and generating pre-metastatic niche in lung Nature Communications, 2021;12(1):474. 2021 [PMID: 33473115] (Bioassay, Human)
• Z Hui, Z Jiang, D Qiao, Z Bo, K Qiyuan, B Shaohua, Y Wenbing, L Wei, L Cheng, L Shuangning, L Zhengyi, L Yingyi Increased expression of LCN2 formed a positive feedback loop with activation of the ERK pathway in human kidney cells during kidney stone formation Scientific Reports, 2020;10(1):21287. 2020 [PMID: 33277533] (Cell Culture, Human)
• K Yahiro, K Ogura, Y Goto, S Iyoda, T Kobayashi, H Takeuchi, M Ohnishi, J Moss Subtilase cytotoxin induces a novel form of Lipocalin 2, which promotes Shiga-toxigenic Escherichia coli survival Sci Rep, 2020;10(1):18943. 2020 [PMID: 33144618] (Cell Culture, Human)
• B Bauvois, E Pramil, L Jondrevill, E Chapiro, C Quiney, K Maloum, SA Susin, F Nguyen-Kha Relation of Neutrophil Gelatinase-Associated Lipocalin Overexpression to the Resistance to Apoptosis of Tumor B Cells in Chronic Lymphocytic Leukemia Cancers (Basel), 2020;12(8):. 2020 [PMID: 32751884] (Bioassay, Human)
• S An, I Raju, B Surenkhuu, JE Kwon, S Gulati, M Karaman, A Pradeep, S Sinha, C Mun, S Jain Neutrophil extracellular traps (NETs) contribute to pathological changes of ocular graft-vs.-host disease (oGVHD) dry eye: Implications for novel biomarkers and therapeutic strategies Ocul Surf, 2019;0(0):. 2019 [PMID: 30965123] (Bioassay, Human)
• Y Zhao, Q Xia, Y Liu, W Bai, Y Yao, J Ding, L Lin, Z Xu, Z Cai, S Wang, E Li, H Xu, B Wu, L Xu, Z Du TCF7L2 and EGR1 synergistic activation of transcription of LCN2 via an ERK1/2-dependent pathway in esophageal squamous cell carcinoma cells Cell. Signal., 2018;0(0):. 2018 [PMID: 30557604] (Bioassay, Human)
• T Parmar, VM Parmar, L Perusek, A Georges, M Takahashi, JW Crabb, A Maeda Lipocalin 2 Plays an Important Role in Regulating Inflammation in Retinal Degeneration J. Immunol., 2018;0(0):. 2018 [PMID: 29602770] (Bioassay, Human)
• C Yan, T Yuanjie, X Zhengqun, C Jiayan, L Kongdan Neutrophil Gelatinase-Associated Lipocalin Attenuates Ischemia/Reperfusion Injury in an In Vitro Model via Autophagy Activation Med. Sci. Monit., 2018;24(0):479-485. 2018 [PMID: 29367586] (Bioassay, Rat)
• S Gomez-Chou, A Swidnicka-, N Badi, M Chavez-Tom, GB Lesinski, T Bekaii-Saa, MR Farren, TA Mace, C Schmidt, Y Liu, D Deng, RF Hwang, L Zhou, TT Moore, D Chatterjee, H Wang, X Leng, RB Arlinghaus, CD Logsdon, Z Cruz-Monse Lipocalin-2 Promotes Pancreatic Ductal Adenocarcinoma by Regulating Inflammation in the Tumor Microenvironment Cancer Res, 2017;0(0):. 2017 [PMID: 28249896] (Bioassay, Human)
• Xiaoye Yan Neutrophil gelatinase-associated lipocalin predicts myocardial dysfunction and mortality in severe sepsis and septic shock Int. J. Cardiol., 2016;0(0):. 2016 [PMID: 27836304]
• Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin A Inhibits Heme Peroxidases PLoS ONE, 2016;11(6):e0156811. 2016 [PMID: 27254317] (Enzyme Assay, Human)
• Cantaluppi V, Dellepiane S, Tamagnone M, Medica D, Figliolini F, Messina M, Manzione A, Gai M, Tognarelli G, Ranghino A, Dolla C, Ferrario S, Tetta C, Segoloni G, Camussi G, Biancone L Neutrophil Gelatinase Associated Lipocalin Is an Early and Accurate Biomarker of Graft Function and Tissue Regeneration in Kidney Transplantation from Extended Criteria Donors. PLoS ONE, 2015;10(6):e0129279. 2015 [PMID: 26125566] (Bioassay, Human)
• Drew B, Hamidi H, Zhou Z, Villanueva C, Krum S, Calkin A, Parks B, Ribas V, Kalajian N, Phun J, Daraei P, Christofk H, Hewitt S, Korach K, Tontonoz P, Lusis A, Slamon D, Hurvitz S, Hevener A Estrogen receptor (ER)alpha-regulated lipocalin 2 expression in adipose tissue links obesity with breast cancer progression. J Biol Chem, 2015;290(9):5566-81. 2015 [PMID: 25468909] (Bioassay, Human)
• La Manna G, Ghinatti G, Tazzari P, Alviano F, Ricci F, Capelli I, Cuna V, Todeschini P, Brunocilla E, Pagliaro P, Bonsi L, Stefoni S Neutrophil gelatinase-associated lipocalin increases HLA-G(+)/FoxP3(+) T-regulatory cell population in an in vitro model of PBMC. PLoS ONE, 2014;9(2):e89497. 2014 [PMID: 24586826] (Bioassay, Human)
• Paton C, Rogowski M, Kozimor A, Stevenson J, Chang H, Cooper J Lipocalin-2 increases fat oxidation in vitro and is correlated with energy expenditure in normal weight but not obese women. Obesity (Silver Spring), 2013;21(12):E640-8. 2013 [PMID: 23640923] (Cell Culture, Mouse)
• Roudkenar MH, Halabian R, Roushandeh AM, Nourani MR, Masroori N, Ebrahimi M, Nikogoftar M, Rouhbakhsh M, Bahmani P, Najafabadi AJ, Shokrgozar MA Lipocalin 2 regulation by thermal stresses: protective role of Lcn2/NGAL against cold and heat stresses. Exp. Cell Res., 2009;315(18):3140-51. 2009 [PMID: 19732769] (Bioassay, Human)
• Pitashny M, Schwartz N, Qing X, Hojaili B, Aranow C, Mackay M, Putterman C Urinary lipocalin-2 is associated with renal disease activity in human lupus nephritis. Arthritis Rheum., 2007;56(6):1894-903. 2007 [PMID: 17530720] (Western Blot, N/A)

Bioinformatics

• Gene Symbol: LCN2
• Uniprot:
  • P80188()