Tlc pei cellulose f plates

Radioactive γ-P32-ATP (PerkinElmer, BLU002Z250UC) was added to 600 μM MgATP (pH = 7.0) solutions at volume ratios of 1:1000-1:300, depending on the lifetime of the radioactive reagent. The total volume of each reaction was 12 μL, including 6 μl of protein from size exclusion chromatography fractions (final concentration ∼0-50 nM for different fractions, peak fractions were used for Rbin-1 and AMPPNP inhibition experiments in Figure 3E), 4 μl FPLC SEC buffer with 0.6 mM Na₂SO₄ and 2 μl MgATP (final concentration = 100 μM). The reactions were then incubated at room temperature for 30 or 60 min before quenching with 12 μl 0.2 M EDTA. 1 μl from each reaction mixture was spotted on to TLC PEI cellulose F plates (Millipore, 105579). The TLC buffer contained 0.15 M formic acid and 0.15 M lithium chloride. The TLC plates were then imaged using the Typhoon Scanner 9400 (GE Healthcare Life Sciences). ImageJ was used to calculate the densitometric ratio of the spots corresponding to radioactive free phosphate and ATP to determine the percent of ATP hydrolyzed.

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).