Typhoon 9410 variable mode imager

Samples after protein precipitation were centrifuged at 6000g for 5 min at 4°C. The supernatant was discarded, and the pellet was washed with ice-cooled methanol twice and air-dried for 10 min. The proteins were resuspended in 1× NuPAGE LDS sample buffer (Invitrogen) with 50 mM DTT and heated at 95°C for 10 min. Samples were then resolved by SDS-PAGE. The labeled proteins were visualized by scanning the gel on a Typhoon 9410 variable mode imager (excitation of 532 nm, emission filter of 580 nm) (GE Healthcare) (23 (link)).

An EcoRI restriction cut site was introduced within the 601 positioning sequence by PCR amplification, 10–15 bp from the left edge of the 601 sequence. Digestions of 150 nM fluorescently labeled [FAM]0N70[LacO-11R,Cy5] nucleosomes were carried out using 2 U/μl of EcoRI-HF (NEB) in 1×CutSmart buffer at 30°C. After pre-incubation of Chd1 (100 nM), nucleosomes (150 nM) and ±LacI (1.2 μM) for 5 min, EcoR1-HF was added for 2 min to digest free DNA, followed by ATP addition to a final concentration of 2.5 mM. Control reactions showed that free DNA was digested within ∼0.5 min. Time points were taken by transferring 1 μl of each 25 μl reaction to 9 μl of quench (SDS-PAGE loading buffer with 40 mM EDTA). Samples (3 μl) were separated by SDS-PAGE (18% acrylamide), visualized using a GE Typhoon 9410 variable mode imager, and quantified using ImageJ (http://imagej.nih.gov/ij/). As previously described for this assay (42 (link)), not all DNA was digested after extended incubation, and all samples were normalized to the same range of nucleosomal DNA that could be cleaved by EcoRI based on samples remodelled with Chd1 in the absence of LacI. Rates were calculated with single exponential fits to data as described above.