Immunological characterization of a newly developed antibody for localization of a beta-keratin in turtle epidermis.
Turtle scutes are made of hard (beta)-keratins. In order to study size and localization of beta-keratins in turtle shell, we produced a rat polyclonal antiserum against a turtle scute beta-keratin of 13-16 kDa, which allowed the immunolocalization of the protein in the epidermis. In immunoblots the antiserum recognized turtle beta-keratins but showed variable cross-reactivity with lizard, snake, and avian beta-keratins. The turtle antiserum appears less cross-reactive than a chicken scale antiserum (Beta-1). In bidimensional immunoblots, three main protein spots at 15-16 kDa with pI at 7.3, 6.8, 6.4, and an unresolved large spot at 40-45 kDa with pI around 5 were more constantly obtained. The latter may result from the aggregation of the smaller beta-keratin protein. The corneous layer of the carapace and plastron of various species of chelonians appeared immunofluorescent. The ultrastructural immunolocalization showed sparse labeling over beta-keratin filaments of cells of the horny layer of both carapace and plastron. The study for the first time shows that the isolated protein band derived from a component of the beta-keratin filaments of the corneous layer of turtles. This antibody can be used for further studies on beta-keratin expression and sequencing in chelonian shell. No labeling was present over other cell organelles or layers of turtle epidermis and it was absent in non-epidermal cells. The specificity for turtle beta-keratin suggests that the antiserum recognizes some epitope/s specific for chelonians beta-keratins, and that it also variably recognizes other reptilian and avian beta-keratins.
Alibardi
Lorenzo
L
Dipartimento di Biologia Evoluzionistica Sperimentale, University of Bologna, 40126 Bologna, Italy. Alibardi@biblio.cib.unibo.it
Toni
Mattia
M
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional; D004817:Epidermis / Q000737:chemistry* / Q000276:immunology; D005455:Fluorescent Antibody Technique; D007106:Immune Sera / Q000737:chemistry; D015151:Immunoblotting; D007150:Immunohistochemistry; D013045:Species Specificity; D014426:Turtles / Q000378:metabolism*; D053800:beta-Keratins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D007106:Immune Sera; D053800:beta-Keratins