Production of anti-peptide antibody of rat brain nitric-oxide synthase

Aim: To raise antibody of rat brain nitric-oxide synthase (bNOS) through immunizing animal with a peptide of bNOS that can represent the holoprotein. Methods: The amino acid sequence for the bNOS was analyzed by GenePro computer program. According to the hydrophilicity, hydrophobicity, antigenicity, and the potentiality to form protein second structures of α-helix, β-sheet and, β-turn, the structure of bNOS was predicted. The peptide 277-287 was selected that was predicted to be in the antigen epitope of bNOS. The peptide was chemically synthesized, coupled to keyhole limpet hemocyanin carrier protein and injected into rabbits to raise antibody. The specificity of the antibody was tested by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blotting. Results: The antibody bound the protein in rat cerebellum extract. In Western blotting, the antibody bound the protein band of 150 kDa in SDS PAGE, and the binding was inhibited by peptide conjugated with carrier protein. In immunohistochemistry, the stain was collocated with the stain in NADPH dehydrogenase histochemistry. Conclusion: The antibody against the peptide recognized the natural bNOS in rat brain, and the peptide 277-287 was located on the surface of bNOS.