| 货号 | 31469 |
| 使用方法 | Recombinant Mononucleosomes (H3.3) - biotinylated are suitable for use in the study of enzyme kinetics, inhibitor screening, and selectivity profiling. HMT Assay Conditions: 2 μg nucleosomes was incubated with different concentration of NSD2-SET (Cat# 31476) in reaction buffer including 50 mM TrisCl, pH 8.6, 0.02% Triton X-100, 2 mM MgCl2, 1 mM TCEP, 50 μM SAM for 3 hours at room temperature. Activity was detected by Western blot. |
| 供应商 | Active Motif |
| 背景 | In vivo, histones are wrapped around by DNA in chromatin. Therefore, nucleosomes are more physiologically relevant substrates than histones and histone-derived peptides for in vitro studies. More importantly, some histone methyltransferases are significantly more active, as well as specific, when using nucleosomal substrates in HMT assays, such as DOT1L and NSD family enzymes. Nucleosomes are also widely used in histone methyltransferase screening assays to identify small molecular inhibitors for drug discovery. |
| 运输条件 | Dry Ice |
| 存放说明 | Recombinant proteins in solution are temperature sensitive and must be stored at -80°C to prevent degradation. Avoid repeated freeze/thaw cycles and keep on ice when not in storage. |
| 外观 | Recombinant Mononucleosomes (H3.3) - biotinylated (20 μg protein + 20 μg DNA) is supplied at a protein concentration of 0.8 μg/μl in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 2 mM DTT and 20% glycerol. |
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Recombinant Mononucleosomes (H3.3) - biotinylated protein gel. 40 µg mononucleosomes were incubated with 15 µl streptavidin beads for 1 h at 4C. Samples were centrifuged at 4C, and supernatant was labelled as free histone octamers. Samples were run on a 12.5% SDS-PAGE gel and stained by Commassie blue. The SDS-PAGE gel result showed that more than 80% biotinylated mononucleosomes were pulled down by streptavidin beads. * indicates streptavidin. | |
Recombinant Mononucleosomes (H3.3) - biotinylated DNA gel. Mononucleosomes were run on an agarose gel and stained with ethidium bromide. Lane 1: 601 DNA which was used for assembly of nucleosome. Lane 2: Intact nucleosome. Intact nucleosomes migrate much higher than free DNA. The agarose gel result showed almost all of the 601 DNA wrapped histone octamers to form nucleosomes. | |
Recombinant Mononucleosomes (H3.3) - biotinylated activity. 2 μg Mononucleosomes were incubated with 0 μg, 0.25 μg, 0.5 μg, 1 μg NSD2-SET (Cat# 31476) in reaction buffer for 3 h at room temperature, respectively. Western blot was used for detecting the generation of reaction products (H3K36me2). Recombinant Histone Octamers was used as a control substrate. The Western blot result shows that nucleosomes are a more suitable substrate for NSD2-SET than histone octamers. |
