dCypher™ Nucleosome K-AcylStat Panel

SKU: 16-9003

dCypher™ K-AcylStat Nucleosome Panel Description: A 96-well plate containing 23 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, 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-AcylStat Nucleosome Panel Formulation: The K-AcylStat panel includes an unmodified nucleosome control and 22 designer nucleosomes (dNucs: single and combinatorial PTMs), either acetylated (ac), butyrylated (bu), or crotonylated (cr) at the indicated lysine sites.

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

dCypher™ K-AcylStat 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 BRD4 BD-1), 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.  16-9003 Datasheet

View full nucleosome panel list.  16-9003 Nucleosome List

16-9003 Panel Layout

dCypher K-AcylStat Nucleosome Panel Plate Layout. The K-AcylStat panel includes an unmodified nucleosome control and 22 designer nucleosomes (dNucs: single and combinatorial PTMs), either acetylated (ac), butyrylated (bu), or crotonylated (cr) at the indicated lysine sites.

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19-6001 Panel Layout

dCypher K-AcylStat Nucleosome Panel Application Data. GST-tagged BRD4 Bromodomain 1 (50 nM, EpiCypher Catalog No. 15-0012) was assayed in AlphaScreen (Perkin Elmer) to measure binding to nucleosomes in the dCypher™ K-AcylStat Nucleosome Panel (10 nM). BRD4 BD-1 showed strong, specific binding to tetra-acetylated histone H4 (H4K5/8/12/16ac), 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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