Bas2500 imaging analyzer

DNA samples were separated using agarose gel electrophoresis. After staining with SYBR Gold, the DNA bands were detected using Gel Doc EZ imager (Bio-Rad) with Image Lab software (Bio-Rad), and its intensities were quantified using ImageJ software (NIH) as a percentage of DNA input, loaded alongside. Protein samples were separated using SDS–PAGE. After staining with CBB, the proteins were detected using Gel Doc EZ imager with Image Lab software, and quantified using ImageJ software as a percentage of protein input, loaded alongside. In ATPase assays, reaction products were separated by thin-layer chromatography and quantified using a BAS2500 imaging analyzer (Fujifilm) with ImageReader software (Fujifilm) and ImageGauge software (Fujifilm). ATP hydrolysis at each time point (0, 15, 30, and 60 min) was calculated from the ratio of inorganic monophosphate and ATP, and the ATPase rates (µM ATP hydrolyzed min⁻¹ per µM RecN) were calculated from the obtained values. The graphs essentially represent the mean ± standard deviation of three independent experiments as indicated in corresponding figure legends.

RecN (1 µM) was incubated at 37 °C in buffer E containing 50 mM NaCl, 2 mM MgCl₂, 5% glycerol, and 2 mM ATP plus [γ³²P]-ATP (PerkinElmer) in the absence or presence of 1.5 nM phiX174-derivative DNA substrates (as a circular or linear molecule). Reaction aliquots were collected at 0, 15, 30, and 60 min, and added to an equal volume of stop buffer (100 mM Tris–HCl at pH 7.5, 100 mM EDTA, and 20 mM ADP). ATP and inorganic phosphate (Pi) in the samples were separated by thin-layer chromatography and quantified using a BAS2500 imaging analyzer (Fujifilm).