In Vitro Fluorescence-Based In-Gel Kinase Assay.

Kinase activity was assessed using a fluorescence-based in-gel phosphoprotein detection assay after SDS-PAGE of the kinase reaction samples. The reactions were carried out in a solution containing 20 mM Hepes (pH 7.5), 200 mM NaCl, 1 mM TCEP, 5 mM MgCl₂, 1 mM ATP, 0.2 µM ctCDK7 or ctCDK7-containing complexes, and 20 µM MBP-scCTD substrate with 26 heptad repeats. Samples were incubated at 37 °C for 1 min and stopped by the addition of SDS sample dye, and then 2 µL of the reaction mix was loaded on a 15% SDS gel. After gel electrophoresis, the gel was stained for phosphoprotein species using the Pro-Q Phosphoprotein Gel Stain Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Signals were detected at an excitation wavelength of 532 nm and an emission wavelength of 605 nm using a PharosFX (Bio-Rad) fluorescence scanner. Signal intensities were quantified using ImageJ (56). All measurements were carried out in technical triplicate and mean values were plotted with their associated SD.