Immunolocalization of intermediate filament proteins using antibodies to synthetic peptides.
Synthetic peptides corresponding to amino acid sequences of amino terminal non-alpha helical domains of human cytokeratin 18 and to low molecular weight human neurofilament subunit were used to obtain monospecific antisera. The results of our immunohistochemical investigations confirmed in general the data previously published on the distribution of cytokeratin 18 in human, rat, and calf tissues. The reactivity of the antiserum was abolished after formalin fixation of specimens. Immunolocalization of the neurofilament subunit using our monospecific antiserum was quite variable from species to species in cells of the central and peripheral nervous systems, and also varied as the result of the tissue fixation procedures. In particular, formalin fixation destroyed the immunoreactivity of the recognized epitope. We discuss the advantages and limits of the use of synthetic peptides as immunogens to produce polyclonal antibodies against intermediate filament proteins, with particular attention to the epitope masking phenomena in cytokeratin polypeptides and the phosphorylation of epitopes in neurofilament subunits.
Sclavo Research Centre, Siena, Italy.
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P
Ruggiero
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R
Petracca
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P
Leoncini
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002417:Cattle; D002490:Central Nervous System / Q000032:analysis / Q000166:cytology / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry / Q000379:methods; D007381:Intermediate Filament Proteins / Q000032:analysis / Q000276:immunology*; D007633:Keratins / Q000032:analysis / Q000276:immunology; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000276:immunology*; D051381:Rats
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D007381:Intermediate Filament Proteins; D010455:Peptides; D007633:Keratins