Chemical synthesis of five tubulin antigenic sequences. Production and characterization of their corresponding anti-tubulin monospecific antibodies.
Five peptides comprising several potential epitopes of alpha and beta-tubulin were synthesized by solid phase methods and purified to homogeneity by HPLC. These are RRNLDIERPTYTN (corresponding to positions 214-226 of the alpha chain of porcine brain tubulin), KDYEEVGVDSVEGE (alpha, 430-443), EGEFSEAREDMAALEKDYEEVGVDSVEGE (alpha, 415-443), RYPGQLNADLRKLAVN (beta, 241-256) and ESNMNDLVSEYQQYQDATAD (beta, 412-431). Appropriate conjugates with carrier proteins rendered all peptides immunogenic, raising antibodies that were cross-reactive with plate-adsorbed tubulin in ELISA. This reaction was completely inhibited by the corresponding free peptides, which indicates that these antibodies react specifically with the regions of alpha and beta-tubulin encompassed by the peptides. The reaction was also fully inhibited by soluble tubulin in a stabilising buffer. In immunoblotting experiments, the anti-peptide antibodies were useful as specific markers for either alpha- or beta-tubulin in brain extracts. The specificity of the anti-peptide antibodies for cellular microtubules was also examined by indirect immunofluorescence experiments.
Andreu
D
D
Rockefeller University, New York, NY.
de la Viña
S
S
Andreu
J M
JM
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D007202:Indicators and Reagents; D014404:Tubulin / Q000138:chemical synthesis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D007202:Indicators and Reagents; D014404:Tubulin