Nucleosome,

Nucleosome, Recombinant Human, H3K4me3 dNuc, Non‑biotinylated

$575.00
SKU: 16-1316
Pack Size: 50 µg

Description

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; H3.2-Q71DI3*; H4-P62805) wrapped by 147 base pairs of 601 positioning sequence DNA. Histone H3 (created by a proprietary semi-synthetic method) contains trimethyl-lysine at position 4. The nucleosome is the basic subunit of chromatin. The 147 bp 601 sequence, identified by Lowary and Widom [1], has high affinity for histone octamers and is useful for nucleosome assembly. The DNA contains a 5’ biotin-TEG group.

*H3K4me3 has a Cys to Ala substitution at position 110.

Validation Data

Figure 1: Protein Gel Data
Coomassie stained PAGE gel of proteins in H3K4me3 dNuc (1 µg) to demonstrate the purity of histones in the preparation. Sizes of molecular weight markers and positions of the core histones (H2A, H2B, H3K4me3 and H4) are indicated.

Figure 2: DNA Gel Data
H3K4me3 dNuc resolved via native PAGE gel and stained with ethidium bromide to visualize DNA. Lane 1: Free DNA (Catalog No. 18-0006; 100 ng). Lane 2: Intact nucleosomes (400 ng).

Figure 3: Mass Spec Data
Synthetic H3K4me3 histone analyzed by high resolution mass spectrometry. Expected mass = 15,267.77 Da. Determined mass = 15,266.47 Da.

Figure 4: Western Blot Data
Western Analysis of H3K4me3 dNuc. Top Panel: Unmodified H3 nucleosomes (Catalog No 16-0009; Lane 1) and H3K4me3 nucleosomes (Lane 2) were probed with an anti-H3K4me3 antibody and analyzed via ECL readout. Only the H3K4me3 sample produced a detectable signal. Bottom Panel: Detail from Coomassie stained gel showing unmodified nucleosomes (Lane 1) and H3K4me3 nucleosomes (Lane 2).

Technical Information

Formulation
H3K4me3 dNuc (27.3 µg protein weight, 50.0 µg DNA + protein) in 10 mM Tris HCl pH 7.5, 25 mM NaCl, 1 mM EDTA, 2 mM DTT, 20% glycerol. MW = 199,250.1.
Storage and Stability
Stable for six (6) months at -80°C from date of receipt. For best results, aliquot and avoid multiple freeze/thaws.

Application Notes

H3K4me3 dNuc is highly purified and suitable for use as a substrate in enzyme screening assays or for effector protein binding experiments.

References

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

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