Tracking ATP Synthesis from ADP via Autoradiography

To investigate dephosphorylation via ATP synthesis, we used KaiC proteins that were expressed as GST fusion proteins and subsequently cleaved off their GST tag. KaiC (3 μM) in ATP synthesis buffer (20 mM Tris-HCl[pH 8], 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl₂, 0.5 mM ATP) was mixed with 0.8 μCi ml⁻¹ [α-³²P]ADP and stored at –20°C or incubated for 2 h at 30°C. As a control, the same experiment was performed in the presence of 0.5 mM ADP. After 20-fold dilution, nucleotides in a 0.5-μl reaction mixture were separated via thin-layer chromatography using TLC PEI cellulose F plates (Merck Millipore) and 1 M LiCl as the solvent. [α-³²P]ADP and [γ-³²P]ATP were separated in parallel to identify the signals corresponding to ADP and ATP, respectively. Dried plates were subjected to autoradiography, and signals were analyzed using a Personal Molecular Imager FX system (Bio-Rad) and ImageLab software (Bio-Rad). For each reaction mixture, the relative intensity of [α-³²P]ATP was calculated as a percentage of all signals in the corresponding lane. Because [α-³²P]ATP was already synthesized during mixing of the samples, the relative ATP intensity measured in the –20°C sample containing 0.5 mM ADP was subtracted for normalization. The principle of this method is based on Egli et al. (12 (link)). A detailed protocol is available on protocols.io (https://doi.org/10.17504/protocols.io.48qgzvw).