The TAP method was applied for purification of cyclin-Cdk1 complexes and Swe1 as described previously for Clb5-TAP-Cdk1 and Clb2-TAP-Cdk1 (Puig et al., 2001; Ubersax et al., 2003 ). 3HA-Cln2-Cdk1 was purified according to published protocols (McCusker et al., 2007 (link)). 6His-tagged recombinant T33-Sic1ΔC constructs and substrates were purified by cobalt affinity chromatography. GST-tagged substrates were purified on glutathione agarose columns.
For the quantitative phosphorylation assays of T33-Sic1ΔC constructs and recombinant substrates, substrate concentrations were kept in the range of 0.5–2 μM (in the linear [S] versus v0 range, several-fold below the estimated KM value), and initial velocity conditions were defined as an initial substrate turnover of up to 10% of the total turnover. For the steady-state peptide kinetics of the Histone peptide PKTPKKAKKL, a similar assay composition was used as for protein substrates, and phosphocellulose paper was used for the quantification of the phosphorylated substrate.
For the western blotting experiments using the Phos-Tag SDS-PAGE, the Sic1ΔC-3HA versions were cloned into vector pRS315 and constitutively expressed under the ADH promoter. The cells were treated for 2.5 hr with 1 μg/ml α factor and released by washing. After the 50 min time point, α factor was readded to collect the cells in the next G1. The cells were lysed by bead beating in lysis buffer containing urea. Blotting of Phos-Tag SDS-PAGE gels was performed using a dry system iBlot (Invitrogen).
For viability assays on galactose plates and for galactose-induced expression time courses, Sic1 mutants were cloned into vector pRS416 under the GAL1 promoter. For flow cytometry experiments, the DNA was stained with propidium iodide and the analysis was performed on a Becton Dickinson BD LSRII flow cytometer.
For the quantitative phosphorylation assays of T33-Sic1ΔC constructs and recombinant substrates, substrate concentrations were kept in the range of 0.5–2 μM (in the linear [S] versus v0 range, several-fold below the estimated KM value), and initial velocity conditions were defined as an initial substrate turnover of up to 10% of the total turnover. For the steady-state peptide kinetics of the Histone peptide PKTPKKAKKL, a similar assay composition was used as for protein substrates, and phosphocellulose paper was used for the quantification of the phosphorylated substrate.
For the western blotting experiments using the Phos-Tag SDS-PAGE, the Sic1ΔC-3HA versions were cloned into vector pRS315 and constitutively expressed under the ADH promoter. The cells were treated for 2.5 hr with 1 μg/ml α factor and released by washing. After the 50 min time point, α factor was readded to collect the cells in the next G1. The cells were lysed by bead beating in lysis buffer containing urea. Blotting of Phos-Tag SDS-PAGE gels was performed using a dry system iBlot (Invitrogen).
For viability assays on galactose plates and for galactose-induced expression time courses, Sic1 mutants were cloned into vector pRS416 under the GAL1 promoter. For flow cytometry experiments, the DNA was stained with propidium iodide and the analysis was performed on a Becton Dickinson BD LSRII flow cytometer.
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