A C-terminal fragment consisting of amino acids 1,350-1,470 from recombinant C. elegans topo-II was amplified with the primers 5'-GCG CGCCCATGGATGTTCGATTCGGACGATGAC-3' and 5'-GCGCGC CTCGAGTCATCTTGGAGCCACAACGAAGC-3'. This fragment was inserted into a bacterial expression vector (pGEX; GE Life Sciences) and expressed in Escherichia coli cells. Purified protein was used for antibody production in rabbit (Institute for Research in Immunology and Cancer core facility). Topo-II antibodies were immunopurified using the same fragment fused to a his tag using standard technologies. A fraction of topo-II and CENP-A antibodies was labeled with DyLight 499 and 649 (DyLight Microscale Labeling kit; Thermo Fisher Scientific). Antibodies were used at concentrations of 10 and 0.33 μg/ml for immunofluorescence and Western blotting, respectively.
top-2