Production of anti-peptide antibody of rat brain nitric-oxide synthase.
Abstract
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 alpha-helix, beta-sheet and
beta-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 limphet 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.
Keywords:
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 alpha-helix, beta-sheet and
beta-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 limphet 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.