dCypher™ Nucleosome K-MetStat & OncoStat Panel

SKU: 16-9002

dCypher™ K-MetStat & OncoStat Nucleosome Panel Description: A 96-well plate containing 24 unique mononucleosomes assembled from recombinant human histones expressed in E. coli (two each of histones H2A, H2B, H3 and H4; accession numbers: H2A-P04908, H2B-O60814, H3.1-P68431/H3.2-Q71DI3/H3.3-P84243, H4-P62805). Single and combinatorial histone post-translational modifications (PTMs) are created using a proprietary synthetic method. The nucleosomes are wrapped by 147 base pairs DNA containing a central 601-positioning sequence (Lowary and Widom 1998 J Mol Biol. (1):19-42) with a 5’ biotin-TEG group.

All nucleosomes in the panel are subjected to EpiCypher’s rigorous quality control metrics, including: ESI-TOF mass spectrometry analysis of the modified proteins; SDS-PAGE to confirm octamer composition and purity; native PAGE to confirm nucleosome assembly and lack of free DNA; and Western blot analysis of the PTM, histone mutation, or histone variant (if applicable). For the full list of nucleosomes in the panel, including individual catalog numbers of full-size (50 µg) products, see the associated excel sheet (link below).

dCypher™ K-MetStat & OncoStat Nucleosome Panel Formulation: Purified recombinant mononucleosomes individually stored in a 96 well plate (1.5 μg nucleosome at 1.5 µM per well) in 5 μL of nucleosome storage buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 25 mM NaCl, 2 mM DTT, & 20% glycerol). MW = ~200,000 Da.

dCypher™ K-MetStat & OncoStat Nucleosome Panel Storage and Stability: Stable for six months at -80°C from date of receipt.

dCypher™ K-MetStat & OncoStat Nucleosome Panel Application Notes: The access to epigenetic diversity in a physiological nucleosome context enables broad end-user applications, including nucleosome binding studies (e.g. chromatin reader binding preferences, see application data below for example using HP1β CHD), enzyme screening assays (e.g. identification of preferred substrates), and antibody specificity testing (e.g. for applications that require antibody specificity in a nucleosome, rather than histone peptide context). The biotin group on the DNA facilitates pull-down experiments. EpiCypher mononucleosomes do not contain free DNA which could alter assayed activities.

View technical datasheet for this product.  16-9002 Datasheet

View full nucleosome panel list.  16-9002 Nucleosome List

16-9002 Panel Layout

dCypher K-MetStat & OncoStat Nucleosome Panel Plate Layout. The K-MetStat & OncoStat panel includes two unmodified nucleosome controls (unmodified H3.1 and H3.3), fifteen designer nucleosomes (dNucs: single PTMs, either mono-, di-, or tri-methylated at the indicated lysine sites), and seven nucleosomes with H3.3 oncogenic mutations (oncoNucs).

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19-6001 Panel Layout

dCypher K-MetStat & OncoStat Nucleosome Panel Application Data. GST-tagged HP1β Chromodomain 1 (30 nM, EpiCypher Catalog No. 15-0058) was assayed in AlphaScreen (Perkin Elmer) to measure binding to nucleosomes in the dCypher™ K-MetStat Nucleosome Panel (10 nM, EpiCypher Catalog No. 16-9002). HP1β showed strong, specific binding to trimethylated lysine 9 of histone H3 (H3K9me3), consistent with its reported binding preference.

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dCypher Panels Frequently Asked Questions

Q: How many assay points of each nucleosome are supplied?
A: This varies according to the sensitivity of the end application. For some context, the supplied format is sufficient to perform >35 AlphaScreen assays,  >30 IP-qPCR experiments, and 3-5 immunoblotting experiments.

Q: How do I store the plate? Can I dilute the nucleosomes?
A: The nucleosomes are stable for 6 months at -80ºC in the supplied format (1.5 μM). Freeze/thaw cycles should be limited (2-3 times). When diluted below 1.5 µM, a carrier protein (e.g. BSA) must be used to supplement the concentration, otherwise, stability is affected. For example, in our SNAP-ChIP product line, nucleosomes are diluted to 0.6 nM in 10 mM sodium cacodylate, pH 7.5, 100 mM NaCl, 1 mM EDTA, 50% glycerol (w/v), 1x Protease Inhibitor cocktail, 100 μg/mL BSA, 10 mM β-mercaptoethanol. In this formulation, the nucleosomes are stable at -20ºC (where they do not freeze) for 6 months.

For applications that may not be compatible with the SNAP-ChIP buffer, EpiCypher routinely dilutes nucleosomes to 40 nM in assay buffer (50 mM Tris pH 7.5, 0.01% Tween-20, 0.01% BSA). Under these conditions the nucleosomes are stable for ~2 weeks at -20ºC.

*Note: extremely high amounts of salt (>650 mM) and ionic detergents (e.g. SDS) will disrupt nucleosome stability.

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