Generation and characterization of novel monoclonal antibodies against human Aurora-A

The mitotic kinase Aurora-A is essential for mitotic progression, including centrosome maturation, mitotic spindle formation, and faithful segregation of chromosomes to daughter cells. Several lines of evidences also suggest that the mammalian aurora kinase family proteins play a role in tumorigenesis. We have previously shown that human Aurora-A was ubiquitinated and negatively regulated by an early mitotic checkpoint protein, Chfr (checkpoint protein with FHA and RING domain). Here, we established several mouse anti-Aurora-A monoclonal antibodies (MAb). GST-tagged human Aurora-A was expressed in BL21 and used as an antigen to immunize mice. Three different hybridomas were obtained and antibodies produced by these hybridomas were analyzed. The results reveal that these antibodies specifically recognize endogenous Aurora-A in both immunoblotting and immunofluroscence experiments. They are useful tools for further analysis of human Aurora-A.