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  • Title: Characterization of antibodies as probes for structural and biochemical studies of tektins from ciliary and flagellar microtubules.
    Author: Linck RW, Goggin MJ, Norrander JM, Steffen W.
    Journal: J Cell Sci; 1987 Nov; 88 ( Pt 4)():453-66. PubMed ID: 3332669.
    Abstract:
    Rabbit antibodies raised and purified against three tektins, proteins of flagellar doublet microtubules from sea-urchin sperm (Lytechinus pictus and Strongylocentrotus purpuratus), were used to study tektin biochemistry and their structural localization. Doublet microtubules were fractionated into tektin filaments and separated by SDS-PAGE into three major tektin polypeptide bands (Mr = 47, 51 and 55 (X 10(3)), which were used to immunize rabbits. Antibodies against each tektin (anti-tektins) were affinity-purified and then characterized by two-dimensional isoelectric focusing/SDS-PAGE immunoblotting and by immunofluorescence microscopy. In two-dimensional immunoblots of 0.5% Sarkosyl-resistant fractions of flagellar microtubules, the antibody against the 55 X 10(3) Mr tektin (anti-55) stained one major polypeptide of 55 X 10(3) Mr and pI 6.9, anti-51 stained two polypeptides of 51 X 10(3) Mr and pI approximately 6.15, and anti-47 stained one major polypeptide of 47 X 10(3) Mr and pI 6.15. The anti-tektins also stained several minor neighbouring polypeptides, which may be isoelectric variants, novel tektins or unrelated proteins. Furthermore, anti-47 crossreacted with the major 55 X 10(3) Mr polypeptide. By immunofluorescence microscopy all three anti-tektins stained methanol-fixed echinoderm sperm flagella and embryonic cilia. In addition, anti-47 and anti-55 stained unfixed, demembranated axonemes. Besides staining axonemes, all anti-tektins labelled the basal body region, and anti-51 labelled the sperm head envelope. These results indicate that the tektins are a complex family of proteins that are components of axonemal microtubules and possibly other cytoplasmic and nuclear structures.
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