title: Characterization of antibodies as probes for structural and biochemical studies of tektins from ciliary and flagellar microtubules.
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.
authors.affiliation: Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis 55455.
authors.forename: R W
authors.initials: RW
authors.lastname: Linck
authors.forename: M J
authors.initials: MJ
authors.lastname: Goggin
authors.forename: J M
authors.initials: JM
authors.lastname: Norrander
authors.forename: W
authors.initials: W
authors.lastname: Steffen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000032:analysis; D005455:Fluorescent Antibody Technique; D007382:Intermediate Filaments / Q000032:analysis; D008856:Microscopy, Fluorescence; D008868:Microtubule Proteins / Q000276:immunology* / Q000378:metabolism; D008870:Microtubules / Q000032:analysis; D012617:Sea Urchins
chemical_list: D000906:Antibodies; D008868:Microtubule Proteins; C044103:tektins