Native gel-shift assays were performed in 20 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mg/mL BSA, 1 mM MgCl2 (link), and 1 mM ADP (or AMPPNP). The concentration of nucleosomes or free DNA was held constant at 50 μM, and Sth1 constructs were added at a range of concentrations to a maximum of 10 μM. The reactions were incubated for 30 min at room temperature, and 10 μL aliquots were loaded onto a 4–20% TBE gel (Bio-Rad) in 0.5 × TBE. The gels were run at 4 °C and 150 V for 3 h (nucleosome) or 45 min (free DNA), and then stained with SYBR Gold (GE Healthcare) and imaged on a Bio-Rad Gel Doc Imager. The bands corresponding to the nucleosome core particle or the 21-bp DNA duplex were quantified densitometrically using the program ImageJ. For each lane, the nucleosome band was quantified and normalized to the no-Sth1 control, and these values were subtracted from 1 to obtain the fraction of nucleosomes bound.
Nucleosome Assembly and Binding Assay
Native gel-shift assays were performed in 20 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mg/mL BSA, 1 mM MgCl2 (link), and 1 mM ADP (or AMPPNP). The concentration of nucleosomes or free DNA was held constant at 50 μM, and Sth1 constructs were added at a range of concentrations to a maximum of 10 μM. The reactions were incubated for 30 min at room temperature, and 10 μL aliquots were loaded onto a 4–20% TBE gel (Bio-Rad) in 0.5 × TBE. The gels were run at 4 °C and 150 V for 3 h (nucleosome) or 45 min (free DNA), and then stained with SYBR Gold (GE Healthcare) and imaged on a Bio-Rad Gel Doc Imager. The bands corresponding to the nucleosome core particle or the 21-bp DNA duplex were quantified densitometrically using the program ImageJ. For each lane, the nucleosome band was quantified and normalized to the no-Sth1 control, and these values were subtracted from 1 to obtain the fraction of nucleosomes bound.
Corresponding Organization :
Other organizations : University of Pennsylvania, University of California, San Diego
Protocol cited in 24 other protocols
Variable analysis
- Concentration of Sth1 constructs
- Fraction of nucleosomes bound
- Concentration of nucleosomes or free DNA (held constant at 50 μM)
- Reaction buffer conditions (20 mM HEPES pH 7.5, 50 mM NaCl, 5% glycerol, 1 mM DTT, 0.1 mg/mL BSA, 1 mM MgCl2, and 1 mM ADP or AMPPNP)
- Incubation time (30 minutes)
- Temperature (room temperature)
- No-Sth1 control (used to normalize the nucleosome band quantification)
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