Monoclonal anti γ tubulin

Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min and then treated with cold methanol for 10 min. The cells were then permeabilized by incubation with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 (PBST) for 15 min. After a 30-min incubation in a blocking solution (PBS containing 3% bovine serum albumin [BSA] and 0.1% Triton X-100), the cells were immunostained with monoclonal anti-Cdc6 (Abcam), monoclonal anti–γ-tubulin (Sigma), polyclonal anti-γ-tubulin (Sigma), monoclonal anti-cyclin E (Santa Cruz), polyconal anti-cyclin A (Santa Cruz), or monoclonal anti-cyclin B (Santa Cruz) antibodies. The anti-CP110 antibodies have been previously described (Chang et al., 2010 (link)). The cells were washed thrice with PBST, incubated with Cy3− or FITC-conjugated anti-rabbit or anti-mouse secondary antibodies, washed thrice with PBST again, and then mounted on glass slides in a mounting medium (Biomeda Corp.), which contained 1 μg/ml 4′,6-diamidino-2-phenylindole DAPI (Vectashield). The cells were observed using an Olympus BX51 microscope.

The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): monoclonal anti-HA, rabbit polyclonal anti-HA and polyclonal anti-Cdc20. Rabbit polyclonal antibodies to Chk1, PChk1 (Ser317), Cdc27, PHistone-H2AX (Ser139), Histone H3 antibodies and mouse monoclonal FLAG antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). The following antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA): monoclonal anti-γ-tubulin, polyclonal α-tubulin, horseradish peroxidase (HRP)-conjugated goat anti-mouse, HRP-conjugated goat anti-rabbit. A human anti-centromere antibody was purchased from Europa Bioproducts Ltd (Cambridge, UK). Monoclonal antibodies to Mad2B, Mad2A, PCNA and c-myc were purchased from BD Transduction Laboratories (Heidelberg, Germany). The following antibodies were purchased from Invitrogen (Carlsbad, CA, USA): Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 rabbit anti-mouse IgG and Alexa Fluor 594 goat anti-human IgG.