Goat anti lcn2 antibody

Neutrophils were plated on glass coverslips coated with 20% fetal calf serum for 10 min at 37°C. The coverslips with attached neutrophils were treated with or without 1 μM formyl-Met-Leu-Phe (fMLP) for 10 min at 37°C and fixed in 2% paraformaldehyde in PBS for 10 min at room temperature [24 (link)]. After permeabilization in 0.1% Triton X-100 in PBS for 5 min, neutrophils were blocked in 10% normal donkey serum (NDS), 0.2% BSA in PBS for 1 h and incubated with mouse anti-PKCδ antibody (1:200 dilution; BD) and goat anti-LCN2 antibody (1:200 dilution; R&D Systems) diluted in PBS containing 2% NDS and 0.2% BSA overnight at 4°C. After three washes with PBS, neutrophils were incubated with the appropriate Donkey fluorochrome-conjugated secondary antibodies (1:200 dilution; Jackson ImmunoResearch) and cover-slipped in mounting media containing DAPI (Vector Labs) to localize nuclei. Images were captured using Zeiss LSM 510 laser confocal microscope.

Dynabeads Protein G (50 µL, Cat# 10007D, Thermo) was washed three times with 1X PBS containing 0.02% Tween-20 (PBST) [57 (link)]. After washing, the Dynabeads were incubated with different concentrations (0, 0.1, 0.5 and 2.5 μg) of LCN2 mAb (Cat# MAB18571, R&D) in 100 µL PBST for 30 min at room temperature. LCN2 mAb bound on the Dynabeads was incubated with 0.1 μg of recombinant mouse LCN2 protein (Cat# 1857-LC, R&D) in 100 µL PBST for 1 h at 4 °C. LCN2 protein-LCN2 mAb complex on the Dynabeads and unbound LCN2 protein in the supernatant were separated by 10% SDS-PAGE, transferred to PVDF membrane, and analyzed by Western blotting. LCN2 mAb bound on the Dynabeads was detected by HRP goat anti-Rat IgG2A (Cat# PA1-84709, Thermo; 1:1000 dilution in Western Antibody Dilution Buffer containing 1× TBS, 0.1% Tween, and 2% nonfat dry milk). The immunoprecipitated LCN2 protein and unbound LCN2 protein in supernatant were detected using goat anti-LCN2 antibody (Cat# AF1857, R&D; 1:1000 dilution in Western Antibody Dilution Buffer) and EasyBlot anti-goat IgG kit (Cat# GTX228910-01, GeneTex, Hsinchu City, Taiwan). Immunoreactive bands were detected using SuperSignal West Pico chemiluminescence substrate (Cat# 34080, Thermo) and imaged using a chemiluminescence imager Celvin S420 and SnapAndGo version1.8 (Biostep Gmbh, Burkhardtsdorf, Germany).