Author: Ramasubramanian Sundaramoorthy; Amanda L. Hughes; Hassane El-Mkami; David Norman; Tom Owen-Hughes
Title: Structure of the chromatin remodelling enzyme Chd1 bound to a ubiquitinylated nucleosome Document date: 2018_3_30
ID: ct2zhauz_55
Snippet: Nucleosomes were reconstituted on Cy3 (me0) and Cy5 (me3) labelled DNA, based on the 601 sequence, with a 47bp extension. Repositioning by Chd1 was performed in 40mM Tris pH7.4, 50mM KCl, 3mM MgCl2, 1mM ATP, 100nM each nucleosome, and 10nM Chd1; 10uL was removed at each time point (T=0, 4, 8, 16, 32, and 64 minutes) , placed on ice, and stopped with the addition of 100ng/uL competitor DNA, 200mM NaCl, and 1.6% sucrose. Repositioned nucleosomes we.....
Document: Nucleosomes were reconstituted on Cy3 (me0) and Cy5 (me3) labelled DNA, based on the 601 sequence, with a 47bp extension. Repositioning by Chd1 was performed in 40mM Tris pH7.4, 50mM KCl, 3mM MgCl2, 1mM ATP, 100nM each nucleosome, and 10nM Chd1; 10uL was removed at each time point (T=0, 4, 8, 16, 32, and 64 minutes) , placed on ice, and stopped with the addition of 100ng/uL competitor DNA, 200mM NaCl, and 1.6% sucrose. Repositioned nucleosomes were run on 6% PAGE/0.2X TBE gels in recirculating 0.2X TBE buffer for 3-4 hours at 300V. The percent of repositioned nucleosomes was analysed using Aida image analysis software. Data were fit to a hyperbola in Sigma Plot, to determine the initial rate of repositioning.
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