Easyblot anti goat igg kit

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).

Dynabeads Protein G (10 µL, Cat# 10007D, Thermo) was incubated with 0.2 μg of LCN2 mAb (Cat# MAB18571, R&D) in 100 µL PBS for 60 min at room temperature [57 (link)]. For the competitive interaction between LCN2 mAb and mouse LCN2 protein, LCN2 mAb bound on the Dynabeads was incubated with 0.06 μg of recombinant mouse LCN2 protein (Cat# 1857-LC, R&D) and mouse LCN2 mAb epitope peptide (YNVTSILVRDQDQGCRYWIRT, synthesized by AnaSpec, Fremont, CA, USA) in 10X, 100X, or 1000X molar ratio of LCN2 protein in 100 µL PBST for 16 h at 4 °C. For the competitive interaction between LCN2 mAb and human LCN2 protein, recombinant human LCN2 protein (Cat# 1757-LC, R&D) and human LCN2 mAb epitope peptide (YNVTSVLFRKKKCDYWIRT, synthesized by AnaSpec) were used. The immunoprecipitated mouse or human LCN2 protein was detected by Western blot using goat anti-mouse LCN2 antibody (Cat# AF1857, R&D; 1:1000 dilution in Western Antibody Dilution Buffer) or goat anti-human LCN2 antibody (Cat# AF1757, R&D; 1:1000 dilution in Western Antibody Dilution Buffer), and an EasyBlot anti-goat IgG kit (Cat# GTX228910-01, GeneTex).