Purification and Interaction of Mitotic Regulators

DNA fragments coding for CIT-K fragments (kinase, CC1, CC2, C1 + PH and CNH), Aurora B, Borealin, INCENP_1-261 and INCENP_525-918 were generated by PCR and cloned into pDEST15 (Invitrogen) to express N-terminal GST-tagged polypeptides in Escherichia coli. The GST-tagged products were then purified using Glutathione Sepharose 4B according to manufacturer's instruction (GE Healthcare). [³⁵S] Methionine-labelled Aurora B, Borealin, INCENP (all three fragments) and CIT-K fragments (kinase, CC2 and CNH) were prepared from corresponding PCR products amplified using primers harbouring a T7 promoter and then in vitro transcribed and translated (IVTT) using the TnT T7 Quick Coupled Transcription/Translation System (Promega) in the presence of [³⁵S] methionine (PerkinElmer). The binding reaction contained 150 mM NaCl and subsequent washes varied from 150 mM to 1 M NaCl. GST pull-down assays were carried out as described [12 (link)].