Nucleosome, Recombinant Human, Acidic Patch Mutant H2AE61A Biotinylated

SKU: 16-0029
Pack Size: 50 µg
  • Species:  Human
  • Source:  E. coli & synthetic DNA
  • Tag:  Biotinylated
  • Molecular Weight:  199,743.9 Da


Recombinant mononucleosomes consist of 147 base pairs of DNA wrapped around an octamer core of histone proteins (two each of H2A, H2B, H3.1, and H4) to form a nucleosome, the basic repeating unit of chromatin. The 147 bp 601 sequence, which contains a 5’ biotin-TEG group, has high affinity for histone octamers and is useful for nucleosome assembly [1]. Histone H2A contains a glutamate-to-alanine (E-to-A) substitution at position 61 (H2AE61A). H2AE61 is among key residues forming a negatively charged region on the nucleosome surface named the “acidic patch”. The acidic patch is a conserved interaction hub for neighboring nucleosomes and nucleosome binding proteins, often via salt bridges with arginine anchors, and is functionally critical in chromatin condensation and chromatin remodeling [2-4]. H2AE61 resides in the alpha2 helix and forms a key salt bridge with H4K16. H2AE61 mediates chromatin binding with factors such as LANA, RCC1, IL-33, SIR3, and HMGN2 [2]. H2AE61A disrupts chromatin remodeling by the ISWI remodeler SMARCA5/SNF2h [4].

Validation Data

Figure 1: Protein gel gata
Coomassie stained PAGE gel of proteins in H2AE61A mononucleosome (1 µg) demonstrates the purity of the histones in the preparation. Sizes of molecular weight markers and positions of the core histones (H2AE61A, H2B, H3 and H4) are indicated.

Figure 2: DNA gel data
H2AE61A mononucleosome resolved by native PAGE and stained with ethidium bromide to visualize DNA. Lane 1: Free DNA (EpiCypher 18-0005; 100 ng). Lane 2: Intact H2AE61A mononucleosomes (400 ng).

Figure 3: Functional binding assay
The presence of acidic patch mutations disrupts LANA peptide binding to recombinant nucleosomes (WT control, EpiCypher 16-0006; H2AE61A, EpiCypher 16-0029; H2AE92K, EpiCypher 16-0030; H2BE105A,E113A, EpiCypher 16-0031). The binding of GST-tagged LANA peptide to biotinylated recombinant nucleosomes was assessed by AlphaLISA assay using Streptavidin Donor Beads and Glutathione Acceptor Beads (PerkinElmer). The presence of H2A acidic patch mutations completely blocks LANA binding, while H2B mutations cause a decrease in LANA binding affinity.

Technical Information

Stable for six months at -80°C from date of receipt. For best results, aliquot and avoid freeze/thaws.
10 mM Tris-HCl pH 7.5, 25 mM NaCl, 1 mM EDTA, 2 mM DTT, 20% glycerol. (27.3 μg protein, 50 μg DNA + protein)

Application Notes

H2AE61A mononucleosome is highly purified and suitable for a variety of applications to test the effect of acidic patch mutation on enzymatic activity or chromatin binding. The biotinylated DNA enables affinity binding applications.

Gene & Protein Information

UniProt ID
H2A - P04908 (alt. names: H2A type 1-B/E, H2A.2, H2A/a, H2A/m)
H2B - O60814 (alt. names: H2B K, HIRA-interacting protein 1)
H3.1 - P68431 (alt. names: H3, H3/a, H3/b, H3/c, H3/d)
H4 - P62805


Background References:
[1] Lowary & Widom J. Mol. Biol. (1998). PMID: 9514715
[2] Kalashnikova et al. J. R. Soc. Interface (2013). PMID: 23446052
[3] Levendosky & Bowman eLife (2019). PMID: 31094676
[4] Gamarra et al. eLife (2018). PMID: 29664398

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