Protein a g plus agarose suspension

Cell lysates (50 µg) were precleared by incubating with 1 µg rabbit/mouse control IgG in 20 µl Protein A/G PLUS-Agarose suspension (Santa Cruz Biotechnology) at 4 °C for 1 h. After centrifugation, the supernatant was incubated with primary antibodies at 4 °C overnight, and then with 20 µl of Protein A/G PLUS-Agarose at 4 °C for 2 h. Immunoprecipitates were collected and rinsed four times with cell lysis buffer. Half of each sample was submitted to the Mass Spectrometry and Biomarker Discovery core at CSMC for interactome analysis, and the remaining half was used to verify the interactome results by western blot.

To investigate the interactions between HsMCM subunits and HsCdc6, HsCdt1, or HsGeminin, Sf9 insect cells expressing each individual protein were lysed in PBS plus 1% Triton X-100 and PIM, and the suspension was centrifuged at 60,000 × g at 4 °C for 30 min. The supernatant from cells expressing each HsMCM subunit was combined with that of cells expressing Myc-HsCdc6, FLAG-HsCdt1, or FLAG-HsGeminin-His₆ or control extract of uninfected Sf9 cells, and was then incubated with epitope-specific agarose beads at 4 °C for 3 h. Afterward, the beads were washed three times with PBS.
To study interactions between HsCdt1 and wild-type or BD mutant HsGeminin, 60 ng of purified FLAG-HsCdt1 was incubated with 40 ng of wild-type or mutant HsGeminin, or control buffer in the presence of 10 μl of protein A/G plus agarose suspension (Santa Cruz Biotechnology, Inc.) and 60 ng of rabbit anti-human Geminin polyclonal antibodies in PBS plus 1 mg/ml BSA and PIM at 4 °C for 3 h. The beads were then washed three times with PBS plus 0.5 m NaCl.
To analyze the precipitated proteins, agarose beads were resuspended in 2× SDS SB, incubated at 100 °C for 10 min, and centrifuged at 16,100 × g for 1 min. After SDS-PAGE of the supernatant, the proteins were transferred to a nitrocellulose membrane and Western blotting analyses with appropriate antibodies were performed.