Purple loading dye without sds

For thermal assays, 2 μl of nucleosome mixtures were mixed with 13 μl Ex80 buffer and incubated at different temperatures (25 (RT), 50.3, 58.9, 65.5 and 69.6°C for 80-Widom nucleosomes and 25 (RT), 50.6, 59.8, 65.0, 70.9 and 75.3°C for Widom and 36-Widom nucleosomes) for 2 h and 4 h, respectively. Afterward 3 μl of Purple Loading Dye without SDS (NEB) was added to the samples and samples were resolved on a 5% polyacrylamide native gel in 0.4× TBE. Gels were analyzed using Typhoon FLA-9500 (GE Healthcare) and MultiGauge v.3.0 (Fujifilm) software.
For ethidium bromide incorporation assays, 2 μl of nucleosome mixtures were added to 13 μl Ex80 buffer containing various ethidium bromide concentrations (up to 1 mM). Samples were incubated at 30°C for 30 min and further processed as described above.

Nucleosomes were assembled using the salt dialysis technique essentially as described previously (27 (link)). A typical assembly reaction (20 μl) contained 250 ng Cy3-labeled DNA (methylated or azidohexynylated) and 250 ng Cy5-labelled DNA (non-modified), 200 ng/μl BSA, 125 ng/μl competitor DNA and different amounts of calf thymus histone octamers. After dialysis 2μl of the assembly reaction was mixed with 8 μl Ex80 buffer (20 mM Tris-HCl pH 7.6, 1 mM MgCl₂, 0.5 mM EGTA, 10% glycerol, 0.05% NP-50, 200 ng/μl BSA, 80 mM KCl) and with Purple Loading Dye without SDS (NEB). Samples were resolved on a 5% polyacrylamide native gel in 0.4× TBE. Gels were analyzed using Typhoon FLA-9500 (GE Healthcare) and MultiGauge v.3.0 (Fujifilm) software.