Recombinant human cdk1 cyclin b1 complex

Recombinant Wee1A proteins (50 nM) were mixed with either Xenopus laevis Cdk1AF-Δ65-cyclin B1 complex (Cdk1AF has non-phosphorylatable residues at Thr 24 (Ala) and Tyr 15 (Phe) and cannot be phosphorylated and inactivated by Wee1-family kinases; Δ65-cyclin B1 is not degraded by the anaphase-promoting complex/cyclosome) (50 nM), Xenopus laevis Cdk1 (wild-type)-Δ65-cyclin B1 complex (300 nM), or recombinant human Cdk1-cyclin B1 complex (1 unit, New England Biolabs) in a kinase assay buffer (5 mM Tris pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mg/mL ovalbumin, 1 μM okadaic acid, 100 μM ATP) at 22°C for 15 min. To test the effect of Cks2, different concentrations of Cks2 (0 to 50 μM) were added. The phosphorylation reactions were stopped by addition of SDS sample buffer.

Bacterial pellets containing recombinant GST-Cks2 proteins were lysed in bacterial lysis buffer (10 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonylfluoride, 1% Triton X-100), clarified and incubated with glutathione-Sepharose (GE Healthcare) in a cold room for 30 min. Recombinant Wee1A proteins were incubated with either recombinant Xenopus laevis Cdk1AF-Δ65-cyclin B1 complex (as described above) or recombinant human Cdk1-cyclin B1 complex (New England Biolabs) in the absence of Cks2. Kinase reactions were performed in a kinase assay buffer (5 mM Tris pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mg/mL ovalbumin, 1 μM okadaic acid, 100 μM ATP) at 22°C for 45 min as des cribed previously with a minor modification (Patra et al., 1999 (link)).
The resulting immobilized GST-Cks2 was incubated with phosphorylated recombinant Wee1A proteins in a binding buffer (50 mM HEPES pH 7.8, 10 mM NaCl) at 4°C for 2 hrs.