Alliance q9 advanced machine

For the P1 nuclease assay, 100 ng pAP205 plasmid was incubated with increasing concentrations of DnaA-6xHis (0.14, 0.54, 1, and 6.3 μM), when required, in P1 buffer (25 mM Hepes-KOH (pH 7.6), 12% (v/v) glycerol, 1 mM CaCl₂, 0.2 mM EDTA, 5 mM ATP, 0.1 mg/ml BSA), at 30°C for 12 min. 0.75 unit of P1 nuclease (Sigma), resuspended in 0.01 M sodium acetate (pH 7.6) was added to the reaction and incubated at 30°C for 5 min. 220 μl of buffer PB (Qiagen) was added and the fragments purified with the minElute PCR Purification Kit (Qiagen), according to manufacturer’s instructions. Digestion with BglII, NotI or ScaI (NEB) of the purified fragments was performed according to manufacturer’s instructions for 1 h at 37°C. Digested samples were resolved on 1% agarose gels in 0.5xTAE (40 mM Tris, 20 mM CH-COOH, 1 mM EDTA PH 8.0) and stained with 0.01 mg/mL ethidium bromide solution afterward. Visualization of the gels was performed on the Alliance Q9 Advanced machine (Uvitec). Images were processed in CorelDraw X7 software. For all experiments at least three independent replicates were performed with various concentrations of DnaA. To quantify the results, background-corrected band intensities were determined using ImageJ, values were normalized against the total signal in a lane in MS Excel, and plotted using GraphPad.

For immunoblotting, proteins were separated on a 12% SDS-PAGE gel and transferred onto nitrocellulose membranes (Amersham), according to the manufacturer’s instructions. The membranes were probed in PBST (PBS pH 7.4, 0.05% (v/v) Tween-20) with a mouse anti-his antibody (1:3000, Invitrogen) and a secondary goat anti-mouse-HRP antibody (1:3000, DAKO) was used. The membranes were visualized using the chemiluminescence detection kit Clarity ECL Western Blotting Substrates (Bio-Rad) in an Alliance Q9 Advanced machine (Uvitec).