Human Lipocalin-2/NGAL ELISA Kit EZ-SetTM (DIY Antibody Pairs)

Lipocalin-2 (LCN2), also known as NGAL, is a protein associated with neutrophil gelatinase.1 The LCN2 gene is located at 9q34 and contains 7 exons.2 The 25-kD LCN2 protein is believed to bind small lipophilic substances such as bacteria-derived lipopolysaccharide (LPS) and formylpeptides and may function as a modulator of inflammation. NGAL tightly binds bacterial catecholate-type ferric siderophores through a cyclically permuted, hybrid electrostatic/cation-pi interaction and is a potent bacteriostatic agent in iron-limiting conditions.3 The primary LCN2 transcript is 3,696 nucleotides long, and the processed transcript is 809 nucleotides long.4 LCN2 expression in adult bone marrow, uterus, prostate, salivary gland, stomach, appendix, colon, trachea, and lung, and in fetal spleen and lung.The standard product used in this kit is recombinant human NGAL, consisting of 178 amino acids with the molecular mass of 22KDa.

Neutrophil gelatinase-associated lipocalin; NGAL;25 kDa alpha-2-microglobulin-related subunit of MMP-9; Lipocalin-2; Oncogene 24p3; Siderocalin LCN2; p25; LCN2; HNL, NGAL

Expressed in bone marrow and in tissues that are prone to exposure to microorganism. High expression is found in bone marrow as well as in uterus, prostate, salivary gland, stomach, appendix, colon, trachea and lung. Not found in the small intestine or peripheral blood leukocytes. .

Intra-Assay Precision Inter-Assay Precision Sample 1 2 3 1 2 3 n 16 16 16 24 24 24 Mean (pg/ml) 256 1510 5422 254 1537 5138 Standard deviation 19.2 81.54 281.94 19.05 84.53 323.69 CV (%) 7.5% 5.4% 5.2% 7.5% 5.5% 6.3% Intra-Assay Precision Inter-Assay Precision Sample 1 2 3 1 2 3 n 16 16 16 24 24 24 Mean (pg/ml) 256 1510 5422 254 1537 5138 Standard deviation 19.2 81.54 281.94 19.05 84.53 323.69 CV (%) 7.5% 5.4% 5.2% 7.5% 5.5% 6.3% Intra-Assay Precision Inter-Assay Precision Intra-Assay Precision Inter-Assay Precision Sample 1 2 3 1 2 3 Sample 1 2 3 1 2 3 n 16 16 16 24 24 24 n 16 16 16 24 24 24 Mean (pg/ml) 256 1510 5422 254 1537 5138 Mean (pg/ml) 256 1510 5422 254 1537 5138 Standard deviation 19.2 81.54 281.94 19.05 84.53 323.69 Standard deviation 19.2 81.54 281.94 19.05 84.53 323.69 CV (%) 7.5% 5.4% 5.2% 7.5% 5.5% 6.3% CV (%) 7.5% 5.4% 5.2% 7.5% 5.5% 6.3%

Iron-trafficking protein involved in multiple processes such as apoptosis, innate immunity and renal development. Binds iron through association with 2,5-dihydroxybenzoic acid (2,5- DHBA), a siderophore that shares structural similarities with bacterial enterobactin, and delivers or removes iron from the cell, depending on the context. Iron-bound form (holo-24p3) is internalized following binding to the SLC22A17 (24p3R) receptor, leading to release of iron and subsequent increase of intracellular iron concentration. In contrast, association of the iron-free form (apo-24p3) with the SLC22A17 (24p3R) receptor is followed by association with an intracellular siderophore, iron chelation and iron transfer to the extracellular medium, thereby reducing intracellular iron concentration. Involved in apoptosis due to interleukin-3 (IL3) deprivation: iron-loaded form increases intracellular iron concentration without promoting apoptosis, while iron-free form decreases intracellular iron levels, inducing expression of the proapoptotic protein BCL2L11/BIM, resulting in apoptosis. Involved in innate immunity, possibly by sequestrating iron, leading to limit bacterial growth. .

Catalog number Description Quantity Storage of opened/reconstituted material EZ0853-CA Rabbit anti- human LCN2 polyclonal antibody (Capture Antibody) 500 μl, 0.4 mg/mL (recommended dilution 1:100) Store undiluted at 4°C for 1 month or at -20°C for 3 months provided this is within the expiration date of the kit. EZ0853-DA Biotinylated goat anti- human LCN2 polyclonal antibody (Detection Antibody) 500 μl, 4.7 μg/mL (recommended dilution 1:100) AR1103 Avidin-Biotin-Peroxidase Complex (ABC) 500 μl (recommended dilution 1:100) EK853-ST Lyophilized recombinant human LCN2 standard 10 ng/tubex3 Discard the standard stock solution after 12 hours at 4°C. May be stored at -20°C for 48 hours provided this is within the expiration date of the kit. Catalog number Description Quantity Storage of opened/reconstituted material EZ0853-CA Rabbit anti- human LCN2 polyclonal antibody (Capture Antibody) 500 μl, 0.4 mg/mL (recommended dilution 1:100) Store undiluted at 4°C for 1 month or at -20°C for 3 months provided this is within the expiration date of the kit. EZ0853-DA Biotinylated goat anti- human LCN2 polyclonal antibody (Detection Antibody) 500 μl, 4.7 μg/mL (recommended dilution 1:100) AR1103 Avidin-Biotin-Peroxidase Complex (ABC) 500 μl (recommended dilution 1:100) EK853-ST Lyophilized recombinant human LCN2 standard 10 ng/tubex3 Discard the standard stock solution after 12 hours at 4°C. May be stored at -20°C for 48 hours provided this is within the expiration date of the kit. Catalog number Description Quantity Storage of opened/reconstituted material Catalog number Description Quantity Storage of opened/reconstituted material EZ0853-CA Rabbit anti- human LCN2 polyclonal antibody (Capture Antibody) 500 μl, 0.4 mg/mL (recommended dilution 1:100) Store undiluted at 4°C for 1 month or at -20°C for 3 months provided this is within the expiration date of the kit. EZ0853-CA Rabbit anti- human LCN2 polyclonal antibody (Capture Antibody) 500 μl, 0.4 mg/mL (recommended dilution 1:100) Store undiluted at 4°C for 1 month or at -20°C for 3 months provided this is within the expiration date of the kit. EZ0853-DA Biotinylated goat anti- human LCN2 polyclonal antibody (Detection Antibody) 500 μl, 4.7 μg/mL (recommended dilution 1:100) EZ0853-DA Biotinylated goat anti- human LCN2 polyclonal antibody (Detection Antibody) 500 μl, 4.7 μg/mL (recommended dilution 1:100) AR1103 Avidin-Biotin-Peroxidase Complex (ABC) 500 μl (recommended dilution 1:100) AR1103 Avidin-Biotin-Peroxidase Complex (ABC) 500 μl (recommended dilution 1:100) EK853-ST Lyophilized recombinant human LCN2 standard 10 ng/tubex3 Discard the standard stock solution after 12 hours at 4°C. May be stored at -20°C for 48 hours provided this is within the expiration date of the kit. EK853-ST Lyophilized recombinant human LCN2 standard 10 ng/tubex3 Discard the standard stock solution after 12 hours at 4°C. May be stored at -20°C for 48 hours provided this is within the expiration date of the kit.

Store at 4°C for 6 months, at -20°C for 12 months. Avoid multiple freeze-thaw cycles (Ships with gel ice, can store for up to 3 days in room temperature. Freeze upon receiving.)

Concentration (pg/ml) 0 156 312 625 1250 2500 5000 10000 O.D. 0.04 0.317 0.479 0.78 1.049 1.588 1.877 2.09 Concentration (pg/ml) 0 156 312 625 1250 2500 5000 10000 O.D. 0.04 0.317 0.479 0.78 1.049 1.588 1.877 2.09 Concentration (pg/ml) 0 156 312 625 1250 2500 5000 10000 Concentration (pg/ml) 0 156 312 625 1250 2500 5000 10000 O.D. 0.04 0.317 0.479 0.78 1.049 1.588 1.877 2.09 O.D. 0.04 0.317 0.479 0.78 1.049 1.588 1.877 2.09

1. Microplate reader in standard size. 2. Automated plate washer. 3. Incubator. 4. Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection. 5. Clean tubes and Eppendorf tubes. 6. 96 well microplate (Cat# AR1100) 7. Plate Sealers. 8. Capture Antibody Diluent: PBS. 9. Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 μm filtered. 10. Substrate Solution: Tetramethylbenzidine (Cat# AR1104) 11. Stop Solution: 2 N H2SO4 (Cat# AR1105) 12. Wash Buffer (PBS and PBS-T) . Recipes: PBS: 8g NaCl, 0.2g KCl, 1.15g Na2HPO4, 0.2g KH2PO4, adjust the total volume to 1 L with distilled water, pH 7.2-7.4, 0.2 μm filtered. PBS-T: 0.1% Tween® 20 in PBS, pH 7.2-7.4. *Item 6 – 12 are included in the EZ Set Accessory Kit (EZA001)