Monoclonal antibodies KN-02 and KN-03 against the heavy chain of kinesin.
The present paper describes two new monoclonal antibodies (MAbs) KN-02 and KN-03 against the heavy chain of conventional kinesin. The kinesin was purified from porcine brain by a combined procedure of ion exchange chromatography, tripolyphosphate-supported microtubule affinity-binding, and gel filtration. Hybridoma cell lines producing antibodies were obtained after immunization of a Balb/c mouse with kinesin and subsequent fusion of the spleen cells with Sp2/0 myeloma cells. The specificity was verified by enzyme-linked immunosorbent assay (ELISA) and further confirmed by immunoblotting and immunoprecipitation analysis. The antibodies recognize different epitopes on the heavy chain of the kinesin molecule as demonstrated by chymotryptic cleavage of kinesin followed by immunoblotting. Differential location of relevant epitopes was also documented by in vitro binding experiments with purified kinesin and taxol-stabilized microtubules. While the KN-03 antibody decorated microtubules, no such staining was observed with KN-02 antibody. The antibodies have a lower affinity to sodium dodecyl sulfate (SDS)-denatured kinesin, but immunofluorescence on fixed cells gave strong dot-like staining characteristic for localization of kinesin on vesicles. The same staining pattern was observed in different cell types. Double-label fluorescence with polyclonal anti-tubulin antibody revealed a co-distribution of stained vesicles with microtubules on the cell periphery. The antibodies KN-02 and KN-03 are therefore valuable tools for localization of kinesins in cells of different tissue origin.
Macůrek
Libor
L
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic.
Dráberová
Eduarda
E
Richterová
Vera
V
Böhm
Konrad J
KJ
Dráber
Pavel
P
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D005260:Female; D005455:Fluorescent Antibody Technique; D016547:Kinesins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008870:Microtubules / Q000276:immunology; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D016547:Kinesins