Laemmli 2x concentrate sample buffer

Total protein was extracted from clarified supernatants using methanol/chloroform extraction method. Samples were then centrifuged at 16,000 x g for 12 minutes at 4°C. The intermediate phase containing the precipitated protein was carefully aspirated and centrifuged for 10 minutes at 4°C. Protein pellets were washed twice in ice-cold methanol. In between washes the resuspended pellets were centrifuged at 16,000 x g for 5 minutes at 4°C. After the final wash, the methanol was carefully removed, and the pellets were allowed to dry. Pellets were re-suspended in Laemmli 2 x concentrate sample buffer (Sigma Aldrich), heated at 100°C for 10 minutes and stored at −80°C until required.

Immunoprecipitations were carried out using Dynabeads Protein G (Invitrogen). Briefly, 25 µL of beads were incubated with 3 µg of KIF11 antibody (Novus; NB500-181; rabbit) for 30 min at room temperature followed by washes and then an overnight incubation at 4 °C with 25 mg of G0 synchronized RPE1 whole cell extract. For analysis of proteins off beads, immunoprecipitated proteins were released from the beads by boiling in Laemmli 2X concentrate sample buffer (Sigma).