Mouse iNOS protein, His tag

Nitric oxide synthase 2, inducible (NOS2) is a member of Nitric oxide synthases (NOSs) family. Nitric oxide synthases (NOSs) are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. NO is an important cellular signaling molecule. It helps modulate vascular tone, insulin secretion, airway tone, and peristalsis, and is involved in angiogenesis and neural development. Besides, Ubiquitin Carboxyl Terminal Hydrolase L5 (UCHL5) has been identified as an interactor of NOS2, thus a binding ELISA assay was conducted to detect the interaction of recombinant mouse NOS2 and recombinant mouse UCHL5. Briefly, NOS2 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to UCHL5-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-NOS2 pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37ºC. Finally, add 50 μl stop solution to the wells and read at 450nm immediately. The binding activity of NOS2 and UCHL5 was in a dose dependent manner.