Phosphorimager typhoon sla9000

Electrophoresis Mobility Shift Assay (EMSA) was carried out with ³²P-labeled DNA in a total volume of 25 µl in 1× EMSA buffer containing 50 mM NaCl, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT, 100 µg/ml acetylated BSA, and 3% glycerol. Reaction mixtures containing ³²P-labeled DNA (with or without unlabeled competitor DNA) and proteins were pre-incubated on ice (unless otherwise indicated) for 30 min. Super-shift experiments were carried out by addition of 0.5–1 µL of specific antibody to the pre-incubated protein-DNA complexes, followed by 20 min incubation on ice (in the presence of a 1000-fold mass excess of unlabeled competitor DNA over ³²P-labeled DNA). Reaction mixtures were finally loaded on pre-run 5% or 8% polyacrylamide gels (29:1 acrylamide/N,N′-methylenebisacrylamide) in 0.5x TBE containing at 200 V (4°C) for 4–6 h. Following the electrophoresis, the gels were dried, and the DNA visualized and quantified on PhosphorImager Typhoon SLA9000 (GE). The dissociation constant K_d was approximated as the total protein concentration at the point of the titration where the fraction of the protein-bound DNA was 0.5 [9] (link), [19] (link).

The assays were performed as previously described [13 (link),21 (link),22 (link)]. Briefly, the ³²P-labeled 123-bp (AvaI ends) or 66-bp (NdeI ends) DNA duplexes (~1 nM) were ligated by T4 DNA ligase (0.05 units, Takara) in the absence or presence of HMGB1 (see the corresponding Figures) at 30^°C for 40min. In some experiments, HMGB1 was pre-incubated with histone H1 or truncated forms of H1 (for exact concentrations of H1 see the Legends to Figures) for 20 min before addition of DNA ligase. Termination of ligation and treatment of samples with Proteinase K was performed as previously described [21 (link)]. Some of the samples were digested (prior to Proteinase K treatment) with 4–20 units of exonuclease III (Promega) at 37^°C for 30 min. Deproteinised DNA samples were then resolved on pre-run 5% polyacrylamide gels in 0.5xTBE buffer (250 V for 4 h at 4^°C), and DNA was visualized and quantified from dried gels on PhosphorImager Typhoon SLA9000 (GE).

The assays were performed as previously described [19] (link), [27] (link). Briefly, the ³²P-labeled 66-bp (NdeI ends) or 123-bp (AvaI ends) DNA duplexes (∼0.2 nM or 2 nM for circularization or DNA end-joining assays, respectively) were ligated by T4 DNA ligase (0.05 units, Takara) in the absence or presence of HMGB1 (typically 6–300 nM) at 30°C for 40 min. Termination of ligation and treatment of samples with Proteinase K was performed as previously reported [19] (link). Some of the samples were digested (before Proteinase K treatment) with 4–20 units of exonuclease III (Promega) at 37°C for 30 min. Protein-free DNA samples were resolved on pre-run 5% polyacrylamide gels in 0.5xTBE buffer (250 V for 4 hs at 4°C) and DNA was visualized and quantified from dried gels on PhosphorImager Typhoon SLA9000 (GE).