Fig. 1. Sequence comparison of vertebrate auroraA kinase non-catalytic domains, recombinant Xenopus auroraA N-terminal protein (Nt-pEg2-(His)6) and antibody specificity. (A) Sequence alignment of vertebrate auroraA N-terminal domains. Xl: Xenopus laevis pEg2, GenBank accession no. Z177207 (Roghi et al., 1998); Mm: Mus musculus AIR1, GenBank accession no. U69106) (Shindo et al., 1998); Hs: Homo sapiens aurora2, GenBank accession no. AF008551 (Shindo et al., 1998). Identical amino acids are shown in grey. (B) Nt-pEg2-(His)6 protein was overexpressed in E. coli and purified by affinity chromatography on a Ni-NTA agarose column. The protein was eluted with 250 mM imidazole and concentrated through a centricon 10. 2 µl (8 µg) were analysed on a 20% SDS-polyacrylamide gel stained with Coomassie Blue. (C) Specificity of 1C1 and 6E3 monoclonal antibodies. 1 µl of a Xenopus egg extract containing about 40 ng of endogenous pEg2 (lanes 2 and 4) or 40 ng of Nt-pEg2-(His)6 purified protein (lanes 1 and 3) were subjected to electrophoresis on a 20% SDS-polyacrylamide gel, transferred on nitrocellulose membrane and probed with 1C1 (lanes 1 and 2) or 6E3 (lanes 3 and 4) monoclonal antibodies (dilution 1/100). The positions of molecular mass (kDa) markers in B and C are shown.
