dCypher™

dCypher™ Nucleosome Full Panel

$2,895.00
SKU: 16-9001

Description

A 96-well plate containing 93 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 199 base pair DNA bearing a 5’ biotin-TEG group and containing a central 601-positioning sequence, identified by Lowary and Widom [1].

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

Figure 2: Chromatin Reader Binding Data
N-terminal 6xHis-tagged HP1β (100 nM, EpiCypher 15-0074) was assayed in AlphaScreen (Perkin Elmer) to measure binding to nucleosomes in the dCypher Nucleosome Panel (10 nM, EpiCypher 16-9001). HP1β showed strong, specific binding to the H3K9me3 dNuc, consistent with its reported binding preference.

Technical Information

Formulation
Recombinant nucleosomes 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

Access to epigenetic diversity in the context of a physiological nucleosome enables broad end-user applications, including nucleosome binding studies (e.g. chromatin reader binding preferences [2-4] see Figure 2), enzyme screening assays (e.g. identification of preferred substrates), and antibody specificity testing (e.g. for applications where histone peptide specificity is an insufficient surrogate). The biotin group on the DNA facilitates a wide variety of applications involving streptavidin capture.

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, such as AlphaScreen, EpiCypher routinely dilutes nucleosomes to 80 nM in assay buffer (20 mM Tris pH 7.5, 0.01% BSA, 0.01% NP-40, 2mM DTT). Under these conditions the nucleosomes are stable for ~2 weeks at -80°C.

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

References

Background References:
[1] Lowary & Widom J. Mol. Biol. (1998). PMID: 9514715
[2] Weinberg et al. Nature (2019). PMID: 31485078
[3] Weinberg et al. Nat Genet. (2021). PMID: 33986537
[4] Dilworth et al. BioRx (2021).

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