ITC studies followed a reported procedure26 (link). The buffer used corresponded to that used in the final protein purification step. Briefly, TAF3PHD and KDM4ATTD: [Tris (50 mM) in water (pH 7.5)]; KDM5APHD3 and BTPFPHD: [Tris (50 mM), NaCl (20 mM) in water (pH 7.5)]; [Tris (25 mM), NaCl (50mM), 1,4-dithiothreitol (1.0 mM) in water (pH 7.5)]. Experiments were conducted using ITC200 automated (GE Healthcare Life Sciences, USA) instrument at 25 °C. Histone peptide titrations were performed with the same reader batches. Solutions of the reader in buffer (25–40 μM) and of the histone H3 peptide (350–600 μM) in buffer were prepared. The prepared solutions were plated into a 96-well plate and inserted into the instrument for analysis. Experiments were performed according to manufacturer’s default settings: Plate pre-rinse syringe clean. A total of 19 injections were performed; each experiment was repeated 3–5 times. Heats of dilution for histone peptides determined in control experiments were subtracted from the titration binding data. Data were analysed with Origin 6.0 (Microcal Inc., Northampton, Massachusetts, USA) and curve fitting with one-site binding mode was applied.
Itc200 automated
Manufactured by GE Healthcare
Sourced in United States
The ITC200 is an automated laboratory instrument designed for the analysis of samples. It features automated sample handling and processing capabilities to perform various analytical tests in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using itc200 automated
1
Histone Peptide Binding Affinity Determination
2
Binding Kinetics of Histone Reader Proteins
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ITC studies followed a reported procedure26 (link). The buffer used corresponded to that used in the final protein purification step. Briefly, TAF3PHD and KDM4ATTD: [Tris (50 mM) in water (pH 7.5)]; KDM5APHD3 and BTPFPHD: [Tris (50 mM), NaCl (20 mM) in water (pH 7.5)]; [Tris (25 mM), NaCl (50 mM), 1,4-dithiothreitol (1.0 mM) in water (pH 7.5)]. Experiments were conducted using ITC200 automated (GE Healthcare Life Sciences, USA) instrument at 25 °C. Histone peptide titrations were performed with the same reader batches. Solutions of the reader in buffer (25–40 µM) and of the histone H3 peptide (350–600 µM) in buffer were prepared. The prepared solutions were plated into a 96-well plate and inserted into the instrument for analysis. Experiments were performed according to manufacturer’s default settings: Plate pre-rinse syringe clean. A total of 19 injections were performed; each experiment was repeated 3–5 times. Heats of dilution for histone peptides determined in control experiments were subtracted from the titration binding data. Data were analysed with Origin 6.0 (Microcal Inc., Northampton, Massachusetts, USA) and curve fitting with one-site binding mode was applied.
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