In vitro kinase assays were performed using 25 μl of kinase reaction. Five nanograms of CDK9/CyclinK (Invitrogen) was incubated with 20 ng baculovirus recombinant full-length MED1 in 10 μM ATP, 40 mM Tris (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 50 μM DTT. DMSO or 1 μM FP was added prior to starting the assay. After 1 h of incubation at 25°C, reactions were stopped by adding an equal volume of 2× SDS sample buffer and heating to 95°C for 5 min.
Cdk9 cyclink
Manufactured by Thermo Fisher Scientific
CDK9/CyclinK is a protein kinase complex that plays a role in the regulation of transcription. It is composed of the catalytic subunit CDK9 and the regulatory subunit Cyclin K. The complex is involved in the phosphorylation of the C-terminal domain of RNA polymerase II, which is important for the initiation and elongation of transcription.
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2 protocols using cdk9 cyclink
1
Kinase Assay of CDK9/CyclinK and MED1
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In vitro kinase assays were performed using 25 μl of kinase reaction. Five nanograms of CDK9/CyclinK (Invitrogen) was incubated with 20 ng baculovirus recombinant full-length MED1 in 10 μM ATP, 40 mM Tris (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 50 μM DTT. DMSO or 1 μM FP was added prior to starting the assay. After 1 h of incubation at 25°C, reactions were stopped by adding an equal volume of 2× SDS sample buffer and heating to 95°C for 5 min.
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In vitro kinase assays were performed using 25 μl of kinase reaction. Five nanograms of CDK9/CyclinK (Invitrogen) was incubated with 20 ng baculovirus recombinant full-length MED1 in 10 μM ATP, 40 mM Tris (pH 7.5), 20 mM MgCl2, 0.1 mg/ml BSA and 50 μM DTT. DMSO or 1 μM FP was added prior to starting the assay. After 1 h of incubation at 25°C, reactions were stopped by adding an equal volume of 2× SDS sample buffer and heating to 95°C for 5 min.
2
Quantifying Kinase Activity Using Isotope-Labeled Formaldehyde
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Titanium dioxide (titania) particle (10 μm diameter) were purchased from GL Sciences (Tokyo, Japan). Thermo-sensitive alkaline phosphatase (TSAP) and modified trypsin were obtained from Promega (Madison, WI). [2H2, 13C]Formaldehyde was purchased from ISOTEC (Miamisburg, OH). Recombinant human protein kinases were from Carna Biosciences (Kobe, Japan), with the exception of KHS1, SGK496, NIK, CDK6/Cyclin D1, CDK9/Cyclin K, ALK1, CDK4/Cyclin D1, BARK1 and MPSK1 (Invitrogen, Carlsbad, CA) and GPRK5, FRAP, DAPK2, PKG1a, STK33, TAO1 and ULT3 (Millipore, Billerica, MA). More details on kinases are shown in Supplementary Table S5 and Supplementary Information. All other reagents were from WAKO Chemicals (Osaka, Japan).
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