Increasing amounts of catalytic domains of Dot1L or yeast Dot1 proteins (50 nM to 1000 nM) were incubated with 12 nM recombinant nucleosomes in EMSA buffer (10 mM Tris pH 7.5, 100 mM NaCl, 2.5% Glycerol and 1 mM DTT) at room temperature for 30 min. The binding reactions were resolved on native polyacrylamide gels (6% PAGE, 0.2 × TBE), stained with SYBR™ Gold (Thermo Fisher), visualized on a Typhoon Trio+ scanner (Molecular Dynamics), and quantified using the program ImageQuant 5.2v (Molecular Dynamics). The amount of Dot1L or yeast Dot1 bound to nucleosomes was determined by measuring the decrease in free nucleosome in each reaction. The background was subtracted from Dot1-free samples. The free DNA was taken under in consideration for the calculation of free nucleosome. The apparent KD and the Hill coefficient for each binding curve were calculated by fitting the specific binding with Hill slope equation using the program Prism 7 (GraphPad). The final parameters were calculated from at least 3 independent experiments. (n≥3/data point). Data were plotted as mean ± s.d.
Imagequant 5.2v
Manufactured by GE Healthcare
The ImageQuant 5.2v is a digital imaging system designed for the analysis of protein and nucleic acid gels. It captures and analyzes images of stained gels, providing quantitative data on the size and intensity of bands or spots.
Automatically generated - may contain errors
2 protocols using imagequant 5.2v
1
Dot1 Binding to Nucleosomes
2
Nucleosome Binding Assay with Orc1 and Sir3
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Increasing amounts of Orc1 or Sir3 proteins were incubated with 50 or 25 nM nucleosomes in reaction buffer (10 mM Tris pH 7.5, 50 mM NaCl, 2.5% glycerol, and 1 mM DTT) at room temperature for 30 min. The binding reactions were resolved on native polyacrylamide gels (6% (for Sir3) or 8% (for Orc1) PAGE, 0.2 X TBE), stained with ethidium bromide (Bio-Rad), visualized on a Typhoon Trio + scanner (Molecular Dynamics), and quantified using the program ImageQuant 5.2 v (Molecular Dynamics). 100 bp Plus DNA ladder (GoldBio) was used to monitor the run. The amount of Orc1 or Sir3 bound to nucleosomes was determined by measuring the decrease in free nucleosome in each reaction. The background was subtracted from BAH-free samples. The free DNA was taking under consideration for the calculation of free nucleosomes. The apparent KD and the Hill coefficient for each binding curve was calculated by fitting the specific binding with the Hill slope equation using the program Prism 7 (GraphPad). The final parameters were calculated from at least three independent experiments (n ≥ 3/data point). Data were plotted as mean ± s.d. A representative EMSAs from each experiment are presented in Supplementary Figs 6 and 7 .
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