Characterization of antibodies as probes for structural and biochemical studies of tektins from ciliary and flagellar microtubules

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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 (× 103)), 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 twodimensional immunoblots of 0·5% Sarkosyl-resistant fractions of flagellar microtubules, the antibody against the 55×103Mr tektin (anti-55) stained one major polypeptide of 55 × 103Mr and pl 6·9, anti-51 stained two polypeptides of 51×103Mr and pl ≈ 6·15, and anti-47 stained one major polypeptide of 47× 103Mr and pl 615. 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 55×103Mr 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.