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Horizontal Tabs
Kinase Assay
Purified CDK complexes at 13 nM concentration were incubated in kinase assay buffer (50 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2, 0.1% v/v NP-40, 1 mM DTT, 20 µM β-glycerophosphate, EDTA-free complete protease inhibitor cocktail) with varying amounts of cold ATP/ [γ-32P]ATP and GST-CTD substrate which contains all 52 heptad repeats of human RNA Pol II C-terminal domain. Reactions were incubated in a circulating 30°C water bath for 15, 30 or 60 minutes. Kinase reactions were stopped with the addition of 6x SDS-PAGE loading dye. Samples were heated at 85°C for 5 minutes and resolved by 4-12% SDS-PAGE. Gels were subsequently dried on 3MM Whatmann paper and imaged with a FujiFilm FLA-7000 scanner. Phosphorylated bands were quantified using FujiFilm MultiGauge™ software. Kinetic parameters (Km values for ATP and GST-CTD) were determined from 2-3 independent experiments. Velocity and Lineweaver-Burke plots were analysed.
Mass spectrometry
Protein masses were determined using an Agilent LC/MSD TOF system with reversed-phase high-performance liquid chromatography coupled to electrospray ionization and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% isopropanol in water with 0.1% formic acid. Spectra were analysed using the MassHunter software (Agilent).
Preparation of full length CDK12/CycK
Full length CDK12 Isoform 1 (NM_016507.2) and CycK1 were cloned into pDONR221 and recombined into bacmids using the Invitrogen Baculovirus Expression System with Gateway Technology. The full length proteins were N-terminally tagged with GST or 6xHis epitopes prior to bacmid generation as per manufacturer’s conditions (Invitrogen pDEST10 and pDEST20 vectors). For expression of the full length CDK12/CycK1 complex, baculoviruses for the epitope-tagged CDK12 and CycK1 were co-infected at a ratio of 4:1 and incubated at 28°C with shaking at 150 rpm. Cells were harvested 48-72 hours post infection. The full length protein complexes were purified by tandem affinity chromatography using either combinations of 6xHis-CDK12/GST-CycK1 or GST-CDK12/6xHis-CycK1. Cell pellets were resuspended in CDK lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 2 mM β-mercaptoethanol, 0.5 mM EDTA, 10 mM β-glycerolphosphate, 0.5 mM sodium orthovanadate, 2 mM NaF, 0.2% v/v NP-40 and EDTA-free complete protease inhibitor cocktail (Roche)). The lysate was incubated on ice for 30 minutes with an additional 0.5 M NaCl and occasional manual mixing. Lysate was then subjected to sonication and clarified by centrifugation. Proteins were captured overnight at 4°C using Ni-NTA agarose (Qiagen) that was pre-equilibrated with CDK equilibrium buffer (10 mM Tris-HCl pH 7.6, 500 mM NaCl, 10% glycerol, EDTA-free complete protease inhibitor cocktail). The Ni-NTA agarose was washed 3x with CDK equilibration buffer and the bead slurry transferred to a disposable column for step-wise elution using CDK equilibration buffer supplemented with 15, 25, 100 and 200 mM imidazole. The eluted protein complexes were dialyzed overnight against CDK activation buffer (12.5 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM EGTA, 5 mM β-glycerolphosphate, 0.5 mM sodium orthovanadate, 2 mM DTT, 0.01% Triton X-100, 10% glycerol, EDTA-free complete protease inhibitor cocktail) to facilitate buffer exchange and removal of imidazole. The protein was concentrated using an Amicon filtration device with a 30 KDa molecular weight cut-off and the retentate was incubated with 500 µM ATP at 30°C for 1 hour to allow auto-activation of the CDK12 kinase. Subsequently, the proteins were further purified using glutathione agarose beads (Pierce). Following overnight batch binding at 4°C, the beads were washed 4x with CDK activation buffer and the bead slurry transferred to a disposable column. Bound protein was eluted in a step-wise fashion with elution buffers (100 mM Tris-HCl pH 7.5, 300 mM NaCl, 1.0 mM EDTA, 0.04% Triton X-100, 4 mM DTT) supplemented with 10 mM and 20 mM glutathione. The purity of the eluted fractions was confirmed by SDS-PAGE or Western blotting before storage at -80°C in 50 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.02% Triton X-100, 2 mM DTT, 50% glycerol. Protein concentration was determined by Bradford assay.
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