For the in vitro HMTase assay, 2 μg substrates were incubated with different enzymes in a methylation reaction buffer [50 mM Tris–HCl (pH 9.0 for GLP/G9a, pH 8.0 for others), 5 mM MgCl2, 4 mM dithiothreitol (DTT), 0.5 mM SAM] at 37°C for the indicated time before analysis by western blotting. Histones H3 and H4 were obtained from New England Biolabs, MA, USA. GST, GST-GLP (containing the Ankyrin and SET domains), GST-SUV39H1, GST-SETD2 and His-SET8 were purified from Escherichia coli. Flag-EZH2 and Flag-DOT1L complexes were purified from HCT116 cells. G9a (containing the Ankyrin and SET domains) was purchased from Cayman Chemical Company, Ann Arbor, MI, USA.
Histones h3 and h4
Manufactured by New England Biolabs
Sourced in United States
Histones H3 and H4 are proteins that are essential components of the nucleosome, the basic unit of chromatin in eukaryotic cells. They play a crucial role in the compaction and organization of DNA within the cell nucleus. These histones are available as individual purified proteins for use in various research applications, such as the study of chromatin structure and function, epigenetic regulation, and protein-DNA interactions.
Automatically generated - may contain errors
2 protocols using histones h3 and h4
1
In vitro HMTase Activity Assay
2
Reconstituted Plasma Membrane Binding Assay
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Egg L-α-phosphatidylcholine (PC), egg L-α phosphatidylethanolamine (PE), and brain (porcine) L-α-phosphatidylserine (PS) in powder form and diacylglycerol (1–2-dioleoyl sn-glycerol, DG) in chloroform were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The powders were resuspended in chloroform, then liposome solutions (PM) consisting solely of PC, PE, and PS were freshly prepared in a 40:40:20 ratio, reflecting those in the plasma membrane. The C1 sensor surface for Biacore X100 was prepared by permanently immobilising a mixture of histones H3 and H4 (New England Biolabs, Ipswich, MA, USA) to cell 2 (active cell) and S100P protein to cell 1 (control cell), as described previously [11 (link)]. Binding curves were generated using PM and DG.
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