dCypher™

dCypher™ Nucleosome Full Panel

$2,495.00
SKU: 16-9001

Description

A 96-well plate containing 77 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 or 187 base pairs of DNA containing a central 601-positioning sequence (1) 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 Documents & Resources section for the associated Excel sheet.

Validation Data

Figure 1: dCypher Nucleosome Full Panel Plate Layout and Key
The full panel includes three unmodified and tailless nucleosome controls, 60 designer nucleosomes (dNucs: single and combinatorial PTMs), seven nucleosomes with H3.3 oncogenic mutations and a wild-type control (oncoNucs), four histone variant nucleosome (vNucs) and 2 nucleosomes with extended 187 bp linker DNA template, either unmodified or methylated (methyl DNA Nucs).

Figure 2: dCypher Nucleosome Full 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™ Nucleosome Panel (10 nM, EpiCypher Catalog No. 16-9001). BRD4 BD-1 showed strong, specific binding to tetra-acetylated histone H4 (H4K5/8/12/16ac), consistent with its reported binding preference.

Technical Information

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.
Storage and Stability
Stable for six months at -80°C from date of receipt. Avoid multiple freeze/thaws.

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.

References

Background References:
(1) Lowary & Widom J. Mol. Biol. (1998) PMID: 9514715

Product References:

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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