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Recombinant Rat Lipocalin-2/NGAL Protein, CF

Catalog #: 3508-LC
3 Citations Datasheet
Catalog # Availability Size / Price Qty
3508-LC-050
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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Rat Lipocalin-2/NGAL Protein, CF Summary

Product Specifications

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.
Source
Mouse myeloma cell line, NS0-derived rat Lipocalin-2/NGAL protein
Gln21-Asn198, with a C-terminal 6-His tag
Accession #
P30152
N-terminal Sequence
Analysis
No results obtained: Gln21 predicted
Predicted Molecular Mass
21 kDa
SDS-PAGE
27 kDa, reducing conditions

Product Datasheets

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3508-LC

Product Datasheet

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

3508-LC

Formulation Lyophilized from a 0.2 μm filtered solution in MES and NaCl.
Reconstitution Reconstitute at 500 μg/mL in sterile 25 mM MES and 150 mM NaCl, pH 6.5.
Shipping The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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 reconstitution.

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 (KD = 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 KD 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); 102137149 (Cynomolgus Monkey)
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; Uterocalin

Citations for Recombinant Rat Lipocalin-2/NGAL Protein, CF

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

3 Citations: Showing 1 - 3
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  1. Lipocalin 2 Suppresses Ocular Inflammation by Inhibiting the Activation of NF-?? Pathway in Endotoxin-Induced Uveitis
    Authors: W Tang, J Ma, R Gu, X Ding, B Lei, X Wang, H Zhuang, G Xu
    Cell. Physiol. Biochem., 2018-03-23;46(1):375-388.
    Species: Rat
    Sample Types: In Vivo
    Applications: In Vivo
  2. Expression and localisation of brain-type organic cation transporter (BOCT/24p3R/LCN2R) in the normal rat hippocampus and after kainate-induced excitotoxicity.
    Authors: Chia W, Tan F, Ong W, Dawe G
    Neurochem Int, 2015-05-21;87(0):43-59.
    Species: Rat
    Sample Types: Whole Cells
    Applications: Bioassay
  3. Iron-related proteins: candidate urine biomarkers in childhood HIV-associated renal diseases.
    Authors: Soler-Garcia AA, Johnson D, Hathout Y, Ray PE
    Clin J Am Soc Nephrol, 2009-03-11;4(4):763-71.
    Applications: ELISA (Standard)

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