The preparation of samples for electrophoresis was performed using the protocols published earlier [35 (link),44 (link)]. Briefly, for the electrophoresis aimed to study the integrity of the EGCG–nucleosome complexes, EGCG (6–100 µM) was added to nucleosomes (60 nM) in 0.01 M buffer for dialysis (10 mM NaCl, 10 mM Tris-HCl (pH 7.9), 0.1% NP40, 0.2 mM EDTA, 5 mM β-ME), incubated for 20 min, mixed with sucrose (final concentration 10%) and loaded into the 4% polyacrylamide gel. Electrophoresis was performed in the HE buffer (10 mM HEPES-NaOH pH 8.0, 0.2 mM EDTA) at +4 °C at 80 V.
In the PARP1-related studies, nucleosomes (2–3 nM) were preincubated with EGCG (0.8–10 µM, 15 min, 25 °C) and further incubated with PARP1 (20 or 50 nM) or with PARP1 and NAD+ (20 μM) in the TB150 buffer (45 min, 25 °C). The probes were subjected to electrophoresis in the 4% polyacrylamide gel (acrylamide: bisacrylamide 59:1; 0.2× TBE buffer) at 120 V for 90 min. Ladder GeneRuler 100 bp (Thermo Fisher Scientific, Waltham, MA, USA) was used as a marker.
The fluorescent imaging of the gels was performed using the Amersham Typhoon RGB imager (GE Healthcare Bio Sciences AB, Uppsala, Sweden).
In the PARP1-related studies, nucleosomes (2–3 nM) were preincubated with EGCG (0.8–10 µM, 15 min, 25 °C) and further incubated with PARP1 (20 or 50 nM) or with PARP1 and NAD+ (20 μM) in the TB150 buffer (45 min, 25 °C). The probes were subjected to electrophoresis in the 4% polyacrylamide gel (acrylamide: bisacrylamide 59:1; 0.2× TBE buffer) at 120 V for 90 min. Ladder GeneRuler 100 bp (Thermo Fisher Scientific, Waltham, MA, USA) was used as a marker.
The fluorescent imaging of the gels was performed using the Amersham Typhoon RGB imager (GE Healthcare Bio Sciences AB, Uppsala, Sweden).
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